tag:blogger.com,1999:blog-37627159044013748372024-03-21T13:08:26.794-07:00S-STEM Scholar Melina Calles's BlogAmanda Chapmanhttp://www.blogger.com/profile/16951554565276475785noreply@blogger.comBlogger18125tag:blogger.com,1999:blog-3762715904401374837.post-8891954355390324392013-04-25T11:55:00.004-07:002013-04-25T11:56:37.203-07:00Poster time !<br />
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;">This week has been a bit crazy because of how little time we
have left in the semester and how much there is still to get done. I am glad to
say that even though the Estrella Conference is right around the corner (next
Thursday) I do not feel too stressed about it, I actually feel pretty calm.
That is my priority as of right now though, I need to get my poster draft done
by tomorrow, and have it ready to go for Monday.<span style="mso-spacerun: yes;"> </span>I am still waiting on the few results I hope
to include in my poster that were done this week.</span></div>
<span style="font-family: Calibri;">For this week I noticed that I had two results of the RFLP
technique using the same enzyme (Ecor1), so I decided to try two different enzymes
and compare them both using the same type of bacteria. The enzyme I used was
Hind III and it had its corresponding buffer. I have provided the protocol for
the RFLP I used with each enzyme.<span style="mso-spacerun: yes;"> </span>Just
to clarify the DNA from the bacteria I used were again extracted from a 24 hour
culture and seemed to have worked great! I wanted to try all seven bacteria for
both protocols even though I knew that I was not successful with a few
extractions (3-4 bacteria did not work) because I have learned that it is sometimes
hard to notice bands using the ultraviolet light because of the concentration
of the sybr green. Below are the results.</span><br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg2vTvycdTCuxSux7ChFthFRun1oPkVXBZpaD3zy7tDslonUYNjXvUiw38sRI-SDO8f85m_tOVqvEC2jGP7jzASCDMCnfZWFbwGTkjmYkYqV5Or_3ZDmw0r2RGoIOSn1wtNVzJjfM9p_qv5/s1600/UVP00125.JPG" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg2vTvycdTCuxSux7ChFthFRun1oPkVXBZpaD3zy7tDslonUYNjXvUiw38sRI-SDO8f85m_tOVqvEC2jGP7jzASCDMCnfZWFbwGTkjmYkYqV5Or_3ZDmw0r2RGoIOSn1wtNVzJjfM9p_qv5/s1600/UVP00125.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Hind III enzyme<br />
</td></tr>
</tbody></table>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;"><strong></strong></span><br />
<span style="font-family: Calibri;"><strong>Bacteria :</strong></span></div>
<ul>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<em>E. coli</em></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<em>P. stuartii</em></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<em>B. subtilis</em></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<em>S. enterica</em></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<em>S. marcescens</em></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<em>S. aureus</em></div>
</li>
<li><table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEieNNReB83X2vFy3mOjym_mkzqAgsYlSzW_obcBEaDfBpRenCdZfHohUfub-zzDauI00VJB-ioQ55YpRoJU52bR2uMJ6qPw88rbYKyexMB_ZeNQiW5fYvVQSWo1LTppYICnRR-3NhQDCvt1/s1600/UVP00124.JPG" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEieNNReB83X2vFy3mOjym_mkzqAgsYlSzW_obcBEaDfBpRenCdZfHohUfub-zzDauI00VJB-ioQ55YpRoJU52bR2uMJ6qPw88rbYKyexMB_ZeNQiW5fYvVQSWo1LTppYICnRR-3NhQDCvt1/s1600/UVP00124.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Ecor 1</td></tr>
</tbody></table>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<em>S. epidermidis</em> </div>
</li>
</ul>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;"><strong>RFLP Protocol</strong></span></div>
<ol>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;">10 ul buffer</span></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;">86 ul DNA</span></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;">4 ul of Ecor1 enzyme or Hind III enzyme</span></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;">Incubated at 37 c degrees for 1 hour</span></div>
</li>
</ol>
<br />
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;">I also decided to run another PCR RAPD protocol on the same
extractions. For the PCR technique I used the DNA which had the best
extractions and used primers 3, 4 and 6. I thought after a few negative results
using primer 7 there would be no use trying it again. Since I ran out of
primers I had to dilute the ones we had in stock. Below I have the calculations
I made for this.<span style="mso-spacerun: yes;"> </span><span style="mso-spacerun: yes;"> </span></span></div>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;"><span style="mso-spacerun: yes;"><strong>Bacteria:</strong></span></span></div>
<ul>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;"><span style="mso-spacerun: yes;"><em>S. marcescens</em></span></span></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;"><span style="mso-spacerun: yes;"><em>S. enterica</em></span></span></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;"><span style="mso-spacerun: yes;"><em>B. subtilis</em></span></span></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<em>S. epidermidis</em></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<em>E. coli</em></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<em>S. aureus</em></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<em>P. stuartii</em></div>
</li>
</ul>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;"><span style="mso-spacerun: yes;"></span></span><strong> Primers</strong> </div>
<ul>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
2 ul primer 3 + 38 ul TE</div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
2ul primer 4 + 38 ul TE</div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
2 ul primer 6 + 38 ul TE</div>
</li>
</ul>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;"><span style="mso-spacerun: yes;"><strong>PCR Protocol</strong> </span></span></div>
<ol>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;"><span style="mso-spacerun: yes;">10 ul master mix</span></span></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;"><span style="mso-spacerun: yes;">5 ul DI H2O</span></span></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;"><span style="mso-spacerun: yes;">4 ul primer (3, 4 or 6)</span></span></div>
</li>
<li><div class="MsoNormal" style="margin: 0in 0in 10pt;">
1 ul DNA <br />
<br />
</div>
</li>
</ol>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
</div>
cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-50428019656239745462013-04-18T14:59:00.000-07:002013-04-18T14:59:44.674-07:00Slowly but surely <!--[if gte mso 9]><xml>
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<br />
<div class="MsoNormal">
I ended my project last week with great extractions, and
with the conclusion that a 24 hour culture is the ideal time I need for
bacteria to grow in, so I can extract DNA from them. What I began to do on
Tuesday was my PCR, <span style="mso-spacerun: yes;"> </span>even though last
time my PCR results showed that primer 7 did not work at all and primer 3,4 and
6 only worked for a few bacteria I decided to work with these four primers once
again. The bacteria I used were the six listed below. My results were awesome
for primer 6! Those are the results I have been aiming for this whole time. I
noticed that primer 3 kind of work but because the gel I ran it in seemed a
little weird I cannot really conclude anything from this. Primer 4 and 7 worked
for only 2 bacteria, each different from the other. I think the reason my PCR
results were better this time around is because for one the practice; the more
I do it the better they should be, and two I made sure all the reagents for
this protocol were on ice.</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<b>DNA</b></div>
<ul>
<li><i>E. coli</i></li>
<li><i>B. subtilis</i></li>
<li><i>S. aureus</i></li>
<li><i>Salmonella</i></li>
<li><i>S. marcescens</i></li>
<li><i>P. stuartii </i><b> </b></li>
</ul>
<b>PCR Protocol</b><br />
<ol>
<li>10 microliters of master mix </li>
<li>
5 microliters of DI H2O</li>
<li>4 microliters of primer (3,4,5,6)</li>
<li>1 microliter of DNA </li>
</ol>
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjNt-L7piJ4YqdBetnBT8eGzHoYW0gdxGsACs7kbvKYkgFd7Jkxk_8mM1uE3iRTWni_inlCCLkU1FAahOttLIGk24YSEuXqCFiUqSmqeikQ4Zjv9Menf3yH9h6cnDhAwiG7lnOZ9LngRE-7/s1600/PCR+Primer+3+with+Extractions+from+4-11-13.JPG" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjNt-L7piJ4YqdBetnBT8eGzHoYW0gdxGsACs7kbvKYkgFd7Jkxk_8mM1uE3iRTWni_inlCCLkU1FAahOttLIGk24YSEuXqCFiUqSmqeikQ4Zjv9Menf3yH9h6cnDhAwiG7lnOZ9LngRE-7/s1600/PCR+Primer+3+with+Extractions+from+4-11-13.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Primer 3</td></tr>
</tbody></table>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh7cR6Ja_Uo81EJymLgNyqIovm_BHUI_CUlhIO7QhR2lxqTsHq_kFYX9SOxh_IJHUK5V0LBX5DGO3LTyXIWDjz37ZCIdti8vQw2ddtQMbvwdQwIvcMaHD3ykf7p1u6RLZmBRm0jmKo9oGHA/s1600/PCR+Primer+4+with+Extractions+from+4-11-13.JPG" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh7cR6Ja_Uo81EJymLgNyqIovm_BHUI_CUlhIO7QhR2lxqTsHq_kFYX9SOxh_IJHUK5V0LBX5DGO3LTyXIWDjz37ZCIdti8vQw2ddtQMbvwdQwIvcMaHD3ykf7p1u6RLZmBRm0jmKo9oGHA/s1600/PCR+Primer+4+with+Extractions+from+4-11-13.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Primer 4</td></tr>
</tbody></table>
<div class="MsoNormal">
<br /></div>
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjJyvfDUk4Hl8fgSPY3gYm-_9y1uhGyGAprnTBDUJyvJGeGxc-jRUg0kdoPuicLkh02A8NP2UQABAxJJUnemXDviW5HsN47V294LrOWnGp45B51u_B67mAU3T1Zzap_DUZeuAwGljIa1fFB/s1600/PCR+Primer+6+with+Extractions+from+4-11-13.JPG" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjJyvfDUk4Hl8fgSPY3gYm-_9y1uhGyGAprnTBDUJyvJGeGxc-jRUg0kdoPuicLkh02A8NP2UQABAxJJUnemXDviW5HsN47V294LrOWnGp45B51u_B67mAU3T1Zzap_DUZeuAwGljIa1fFB/s1600/PCR+Primer+6+with+Extractions+from+4-11-13.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Primer 6</td></tr>
</tbody></table>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj3vpXveZrZA_EmfzvKCjW5a79NDHdHLGM_pvxQLr3vCmDbUHJyVO5rrmOOuFnWRoMAU2rNz0Ld8ejJDgvJ_GcVKqSaaTmyXFLhh8-8d6c4ks0eMacr5nL9YwEuWdY0LHADtyJcIMUKrKVT/s1600/PCR++Primer+7+with+Extractios+from+4-11-13.JPG" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj3vpXveZrZA_EmfzvKCjW5a79NDHdHLGM_pvxQLr3vCmDbUHJyVO5rrmOOuFnWRoMAU2rNz0Ld8ejJDgvJ_GcVKqSaaTmyXFLhh8-8d6c4ks0eMacr5nL9YwEuWdY0LHADtyJcIMUKrKVT/s1600/PCR++Primer+7+with+Extractios+from+4-11-13.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Primer 7</td></tr>
</tbody></table>
<div class="MsoNormal">
<br /></div>
<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgUHaLwRS8APGJR5shu4-q8LAm9Hizp6_AlPytR8csdyuNmN30Z0xqfuYLwuXRiq8bVlCxFt78ibnBn6NKx3WMVBQ57ietGDOiW8Uk3GGrczkGbjx_HGNu3bLG14ocy1Zk8DjGHcr0ez_q_/s1600/UVP00111.JPG" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgUHaLwRS8APGJR5shu4-q8LAm9Hizp6_AlPytR8csdyuNmN30Z0xqfuYLwuXRiq8bVlCxFt78ibnBn6NKx3WMVBQ57ietGDOiW8Uk3GGrczkGbjx_HGNu3bLG14ocy1Zk8DjGHcr0ez_q_/s1600/UVP00111.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">RFLP Results using DNA extractions from 4-11-13</td><td class="tr-caption" style="text-align: center;"><br /></td></tr>
</tbody></table>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<b>RFLP Protocol</b></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
1. 10 microliters of 1 x buffer</div>
<div class="MsoNormal">
2. 88 microliters of DNA</div>
<div class="MsoNormal">
3. 2 microliters of Ecor 1 enzyme</div>
<br />
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
For the RFLP I used the same DNA extractions that were done
on 4-11-13, and the same protocol I used for the RFLP done on 4-4-13. My
results were what we would call sloppy, because you can see the DNA and you can
see where the markers are but the DNA is just smeared all down the lane. We are
now trying to find a way to lower the concentration of DNA because we think the
reason we keep getting bright lanes all the way down is because the DNA
concentrations are high.</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<a href="http://upload.wikimedia.org/wikipedia/commons/5/51/Cephalocereus_senilis.jpg" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" src="http://upload.wikimedia.org/wikipedia/commons/5/51/Cephalocereus_senilis.jpg" height="200" width="150" /></a>I also want to add that on Friday I was able to experience
the wonderful Desert Botanical Garden, it was awesome and I got to learn the
plants we have here in Arizona. Thanks to my advisors, Dijana, and Schampel I
was able to learn so much about these plants and reinforce some information I
was introduced to in my biology classes. I also want to share with you the
plant the cactus that made my day at this garden. This cactus is called the old
man cactus. </div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<br />
<br />
<br />
<br />cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-54542596163210276582013-04-12T01:00:00.000-07:002013-04-12T01:00:36.131-07:00Another week down, about three to go! <br />
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";">I
started off my week with great news, I found out I will be able to participate
in the Estrella Mountain Conference on May 2<sup>nd</sup>. <o:p></o:p></span></div>
<br />
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";">Last
week I talked about my extractions and my RFLP (restriction fragment length
polymorphism), I mentioned how my results were not the clearest and you could
see at the most 1 band in the gel when in reality I should be getting much more
than that. I will continue practicing this technique next week.<o:p></o:p></span></div>
<br />
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";">If
you remember I mentioned before that my project will consist of the RFLP
technique but also the PCR technique. All of last week the biology classes
needed the PCR machine for their own labs and I did not have much access to it.
This week however I took advantage of the machine being returned and that was
the first thing I ran my extractions in. My protocol for the PCR technique was
the same as the one I used before. <span style="mso-spacerun: yes;"> </span>I had
to dilute the primers that I worked with, and I have included my calculations
for the primers. I made sure to use the three best extractions that worked from
my previous results. The three bacteria I worked on for this PCR were<o:p></o:p></span></div>
<ul>
<li><span style="color: black; font-family: Symbol; font-size: 12pt; line-height: 115%; mso-bidi-font-family: Symbol; mso-fareast-font-family: Symbol;"><span style="mso-list: Ignore;"><span style="font-size-adjust: none; font-stretch: normal; font: 7pt/normal "Times New Roman";">
</span></span></span><!--[endif]--><i style="mso-bidi-font-style: normal;"><span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";">S. aureus</span></i><span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";"> from the extractions
done on 3-28-13</span></li>
<li><i style="mso-bidi-font-style: normal;"><span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";">S. marcescens</span></i><span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";"> from the extraction
done on 3-28-13</span></li>
<li><i style="mso-bidi-font-style: normal;"><span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";">P. stuartii</span></i><span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";"> from the extraction
done on 4-2-13</span></li>
</ul>
<br />
<div class="MsoNormal" style="line-height: normal; margin: 0in 0in 0pt;">
<span style="font-family: Calibri;"><strong>Primers PCR Protocol</strong></span></div>
<br />
<div class="MsoNormal" style="line-height: normal; margin: 0in 0in 0pt;">
<span style="font-family: Calibri;">2ul of primer 3 + 38ul of TE H2O= Primer 3 <span style="mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin;"><span style="mso-list: Ignore;"><span style="font-family: Calibri;">1.</span><span style="font-size-adjust: none; font-stretch: normal; font: 7pt/normal "Times New Roman";"> </span></span></span><!--[endif]--><span style="font-family: Calibri;">10ul of master mix</span></span></div>
<br />
<div class="MsoNormal" style="line-height: normal; margin: 0in 0in 0pt;">
<span style="font-family: Calibri;">2ul of primer 4 + 38ul of TE H2O= Primer 4 <span style="mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin;"><span style="mso-list: Ignore;"><span style="font-family: Calibri;">2.</span><span style="font-size-adjust: none; font-stretch: normal; font: 7pt/normal "Times New Roman";"> </span></span></span><!--[endif]--><span style="font-family: Calibri;">5 ul of DI H2O</span></span></div>
<br />
<div class="MsoNormal" style="line-height: normal; margin: 0in 0in 0pt;">
<span style="font-family: Calibri;">2ul of primer 6 + 38ul of TE H2O= Primer 6 3. 4ul of Primer (3,4,6,7)</span></div>
<br />
<div class="MsoNormal" style="line-height: normal; margin: 0in 0in 0pt;">
<span style="font-family: Calibri;">2ul of primer 7 + 38ul of TE H2O= Primer 7 <span style="mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin;"><span style="mso-list: Ignore;"><span style="font-family: Calibri;">4.</span><span style="font-size-adjust: none; font-stretch: normal; font: 7pt/normal "Times New Roman";"> </span></span></span><!--[endif]--><span style="font-family: Calibri;">1ul of DNA</span></span></div>
<br />
<br />
<div class="MsoListParagraphCxSpLast" style="margin: 0in 0in 10pt 38.25pt; mso-add-space: auto; mso-list: l1 level1 lfo2; text-indent: -0.25in;">
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; height: 259px; text-align: right; width: 212px;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhi7CN8JuVWQkJY7CEZwTwGvJMdvkhnZv_QasJ0y5AXoe1BBxqt-toJlgJa1flXvvNXziZ660HpxXtYF5q1SxIZtOL9ZeItm5TJibp54AoCrx8PYcRT8PMAZz675WAw_iGjVSDQ3fmc0v1d/s1600/Primer+6+and+7.JPG" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhi7CN8JuVWQkJY7CEZwTwGvJMdvkhnZv_QasJ0y5AXoe1BBxqt-toJlgJa1flXvvNXziZ660HpxXtYF5q1SxIZtOL9ZeItm5TJibp54AoCrx8PYcRT8PMAZz675WAw_iGjVSDQ3fmc0v1d/s1600/Primer+6+and+7.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">PCR Results using Primer 6 & 7<br />
Ruler<br />
<em>S. marcescens</em> primer 6<br />
<em>S. aureus</em> primer 6<br />
<em>P. stuartii </em>primer 6<br />
<em>S. marcescens</em> primer7<br />
<em>S. aurues</em> primer 7<br />
<em>P. stuartii</em> primer 7<br />
</td></tr>
</tbody></table>
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjgHLGTiwn0YtfJPYICqDCKq1YKx9sQVxY6TGlZzpjxil_io0szACzVVqeoTtyKhis51ChGZOLjgwq5nwzH8yAlfdn2AHg_htQD2ZPcJg1q-hVMWhZHFvw2WDg1ya_1-Nat_B3wNnhGkGNd/s1600/Primer+3+and+4.JPG" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjgHLGTiwn0YtfJPYICqDCKq1YKx9sQVxY6TGlZzpjxil_io0szACzVVqeoTtyKhis51ChGZOLjgwq5nwzH8yAlfdn2AHg_htQD2ZPcJg1q-hVMWhZHFvw2WDg1ya_1-Nat_B3wNnhGkGNd/s1600/Primer+3+and+4.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">PCR Results using primer 3 & 4<br />
Ruler<br />
<em>S. marcescens</em> primer 3<br />
<em>S. aureus</em> primer 3<br />
<em>P. stuartii</em> primer 3<br />
<em>S. marcescens</em> primer 4<br />
<em>S. aureus</em> primer 4<br />
<em>P. stuartii</em> primer 4<br />
</td></tr>
</tbody></table>
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";"></span> </div>
<div class="MsoListParagraphCxSpLast" style="margin: 0in 0in 10pt 38.25pt; mso-add-space: auto; mso-list: l1 level1 lfo2; text-indent: -0.25in;">
</div>
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";"></span><br />
<br />
<div class="MsoListParagraphCxSpLast" style="margin: 0in 0in 10pt 38.25pt; mso-add-space: auto; mso-list: l1 level1 lfo2; text-indent: -0.25in;">
</div>
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";">
</span><br />
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
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have included a picture of my results. I had some trouble with the
electrophoresis gel, the machine would not let me run the gel and it would keep
turning off, I decided to unplug it for a few minutes and start again and it
worked!</span><span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";"><br style="mso-special-character: line-break;" />
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="clear: left; float: left; margin-bottom: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi4N-pdJW-TSsrCta-WyLiAKZzODKOs88PS268PPdWiDHkzkm64RgKPmNoja8jnuhTFHgFeVjuCqq0KJe_NFKFQ53dIE1XJD3sVB5RdFO34LL1LMRtFS1dSXucwEeAP5Xaiz64JBCbI2B-a/s1600/Extractions+4-11-13.JPG" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi4N-pdJW-TSsrCta-WyLiAKZzODKOs88PS268PPdWiDHkzkm64RgKPmNoja8jnuhTFHgFeVjuCqq0KJe_NFKFQ53dIE1XJD3sVB5RdFO34LL1LMRtFS1dSXucwEeAP5Xaiz64JBCbI2B-a/s1600/Extractions+4-11-13.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">QuickExtract Kit Extractions<br />
Ruler<br />
<em>E. Coli</em><br />
<em>B. subtilis</em><br />
<em>S. aureus</em><br />
<em>S. marcescens</em><br />
<em>P. stuartii</em><br />
<em>Salmonella</em><br />
<em>S. epidermidis</em></td></tr>
</tbody></table>
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";"><span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";">
</span><span style="color: black; font-family: "Times New Roman","serif"; font-size: 12pt; line-height: 115%; mso-fareast-font-family: "Times New Roman";">I
would like mention that Matt and discussed other reasons why I am having
trouble with my extractions, we think it is because of how old the cultures are
that I work with before I extract them. We concluded at from now on I should
use cultures that are only 24 hours old and see if that works better. Today, I
began new extractions with the fresh 24 hour culture. Here were my results! You
can see that the bands are brighter and they all show DNA.<o:p></o:p></span></span></div>
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cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-60238218257110481262013-04-04T12:27:00.003-07:002013-04-04T12:27:59.422-07:00Ok so about two weeks ago I think you can recall that I was missing my Extraction Kit so I began extracting DNA from <em>E.coli </em>using a different protocol. My extraction was successful, but there was a small amount of it. Anyway, when I finished the extraction Josh asked me to try this new RFLP project in what is now my actual project, on <em>E.coli. </em>This RFLP stands for restriction fragment length polymorphism, and is a technique that basically cuts the DNA sequence at specific spots. Certain enzymes in this case restriction enzymes are the ones who look for the same sequence repeating throughout the entire DNA and pretty much just "break" it, or digests it at that spot. I check my results for this project by running my DNA in an electrophoresis gel. When I first tried it with the<em> E. coli</em> I had too small of an amount to believe that anything could appear and figure 1 shows this. Below are the amounts of DNA, enzymes, H2O, and buffers that I used. It was not successful and one of the reasons why is because later on Matt and Josh realized the buffer and enzymes I used were about 5-6 years old! Of course this wasn't going to work! <br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgZ8uqS8K1KAZdlqZmM-_rOwFyJaUZksUksVIujo-e6xUFLcO5LuI1Xdw7GLFWxpVm5NbqLR2-e2CKhBy2sVeM57JZmCIUEvKByWEh1P3xoOgvqZN-VG31vkheGKmpFvcSd6i1VsGS6HS5J/s1600/3-21-13+RFLP.JPG" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgZ8uqS8K1KAZdlqZmM-_rOwFyJaUZksUksVIujo-e6xUFLcO5LuI1Xdw7GLFWxpVm5NbqLR2-e2CKhBy2sVeM57JZmCIUEvKByWEh1P3xoOgvqZN-VG31vkheGKmpFvcSd6i1VsGS6HS5J/s1600/3-21-13+RFLP.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1<br />
RFLP using <em>E.coli</em> <br />
Ruler, A, B, C </td></tr>
</tbody></table>
<br />
<table border="0" cellpadding="0" cellspacing="0" style="border-collapse: collapse; width: 301px;">
<colgroup><col style="mso-width-alt: 1572; mso-width-source: userset; width: 32pt;" width="43"></col>
<col style="mso-width-alt: 2669; mso-width-source: userset; width: 55pt;" width="73"></col>
<col style="mso-width-alt: 1389; mso-width-source: userset; width: 29pt;" width="38"></col>
<col style="mso-width-alt: 1426; mso-width-source: userset; width: 29pt;" width="39"></col>
<col style="mso-width-alt: 2377; mso-width-source: userset; width: 49pt;" width="65"></col>
<col style="mso-width-alt: 1572; mso-width-source: userset; width: 32pt;" width="43"></col>
<tbody>
<tr height="20" style="height: 15pt;">
<td height="20" style="background-color: transparent; border: 0px black; height: 15pt; width: 32pt;" width="43"><span style="font-family: Calibri;">Tube</span></td>
<td style="background-color: transparent; border: 0px black; width: 55pt;" width="73"><span style="font-family: Calibri;">10x buffer</span></td>
<td style="background-color: transparent; border: 0px black; width: 29pt;" width="38"><span style="font-family: Calibri;">DNA</span></td>
<td style="background-color: transparent; border: 0px black; width: 29pt;" width="39"><span style="font-family: Calibri;">H2O</span></td>
<td style="background-color: transparent; border: 0px black; width: 49pt;" width="65"><span style="font-family: Calibri;">Hind 111</span></td>
<td style="background-color: transparent; border: 0px black; width: 32pt;" width="43"><span style="font-family: Calibri;">Ecor 1</span></td>
</tr>
<tr height="20" style="height: 15pt;">
<td height="20" style="background-color: transparent; border: 0px black; height: 15pt;"><span style="font-family: Calibri;">A</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">2ul</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">1ul</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">17ul</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">0ul</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">0ul</span></td>
</tr>
<tr height="20" style="height: 15pt;">
<td height="20" style="background-color: transparent; border: 0px black; height: 15pt;"><span style="font-family: Calibri;">B</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">10ul</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">50ul</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">32ul</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">8ul</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">0ul</span></td>
</tr>
<tr height="20" style="height: 15pt;">
<td height="20" style="background-color: transparent; border: 0px black; height: 15pt;"><span style="font-family: Calibri;">C</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">2ul</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">2ul</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">17ul</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">0ul</span></td>
<td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;">1ul<span style="mso-spacerun: yes;"> </span></span></td>
</tr>
</tbody></colgroup></table>
<table border="0" cellpadding="0" cellspacing="0" style="border-collapse: collapse; width: 396px;"><colgroup><col style="width: 48pt;" width="64"></col><col style="mso-width-alt: 2779; mso-width-source: userset; width: 57pt;" width="76"></col><col span="4" style="width: 48pt;" width="64"></col><tbody>
<tr height="20" style="height: 15pt;"><td height="20" style="background-color: transparent; border: 0px black; height: 15pt; width: 48pt;" width="64"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black; width: 57pt;" width="76"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black; width: 48pt;" width="64"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black; width: 48pt;" width="64"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black; width: 48pt;" width="64"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black; width: 48pt;" width="64"><span style="font-family: Calibri;"></span></td></tr>
<tr height="20" style="height: 15pt;"><td height="20" style="background-color: transparent; border: 0px black; height: 15pt;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td></tr>
<tr height="20" style="height: 15pt;"><td height="20" style="background-color: transparent; border: 0px black; height: 15pt;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td></tr>
<tr height="20" style="height: 15pt;"><td height="20" style="background-color: transparent; border: 0px black; height: 15pt;"></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"></span></td><td style="background-color: transparent; border: 0px black;"><span style="font-family: Calibri;"><span style="mso-spacerun: yes;"> </span></span></td></tr>
</tbody></colgroup></table>
This week some new enzymes and buffers came in and I was able to try the RFLP technique for the first time. I had to make a few calculations to figure out how much of each (buffer, enzyme, DNA) I would need for a total of 100ul of volume. My conclusions were the following<br />
<ol>
<li>10ul of 1x buffer</li>
<li>88ul of DNA</li>
<li>2ul of Ecor1 enzyme </li>
</ol>
I added them in this order as well, because if you add the enzyme before you have everything you want in the test tube than this enzyme will actually start to react with itself and whatever is in the test tube. After added all these reagents together, I placed them in a water bath that was set at 37 degrees C for an hour. After that I followed the electrophoresis gel protocol with 1% agarose.<br />
<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi32tOC_u8HhzcS5JVvYSLOddl4AwJH_53L2ZjeE3e2XmUwdI9r5NQv0rd5M5r6okz7RCZGuSCmkiR35AwLmQSRfKHfEK4XBFBOqnCic_BDyVV72gZumiZPGOZ3WlEXt-N5fwBsl_o2WrWu/s1600/UVP00102.JPG" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi32tOC_u8HhzcS5JVvYSLOddl4AwJH_53L2ZjeE3e2XmUwdI9r5NQv0rd5M5r6okz7RCZGuSCmkiR35AwLmQSRfKHfEK4XBFBOqnCic_BDyVV72gZumiZPGOZ3WlEXt-N5fwBsl_o2WrWu/s1600/UVP00102.JPG" height="150" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">RFLP <br />
Ruler<br />
<em>S.</em> <em>marcescens</em> (3-38-13) <br />
<em>S. marcescens</em> (3-26-13)<br />
<em>S. aureus</em> (3-38-13)<br />
<em>P. stuartii</em> (4-2-13)<br />
<br />
</td></tr>
</tbody></table>
<ol>
<li>1ul sybr green</li>
<li>2ul orange loading dye </li>
<li>8ul of DNA</li>
</ol>
<br />
<ol>
<li>9ul molecular weight ruler</li>
<li>1ul sybr green</li>
</ol>
<br />
I added them in the order you see. The gel ran for 35 minutes at 120 volts in dark conditions. You can see from my results that there was some digesting going on but not like I was expecting. <br />
<br />
<br />
cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-10749401183760860812013-03-28T14:24:00.001-07:002013-03-28T14:26:17.506-07:00Extractions ! This week started off in a productive and interesting way. The reason I say
this is because on Monday each of us had the chance to apply to another conference which will be taking place at Estrella Mountain Community
College. Since the deadline was Monday we all had to revise our abstracts to submit them and have the chance to
participate in this conference. <br />
<br />
I am excited because my project changed a little bit, I
will not be focusing on the best way to extract DNA anymore because I already
found out what the best way is which was using the Epicentre QuickExtract kit,
so what my project will focus on now is on the amount of successful PCR and RFLP
results I will get from these extractions. In addition to this I will be
extracting more bacteria then what I was working with.<br />
<br />
On Tuesday I extracted DNA from the seven cultures I made last
week. I noticed that the <i style="mso-bidi-font-style: normal;">S. epidermidis</i> bacteria did not grow so what Matt advise me was to
set it aside and create another culture for it because it was obvious I was not
going to be able to extract anything from it so I only extracted six bacteria.
After my extractions were done I ran them in a .5 % agarose gel, but did not have great results so then I ran a new
electrophoresis gel with 1% agarose to see if my results would
change. I saw better results but I still lacked about three bacteria. Also I noted that instead of using normal 1x sybr green dye for running these gels I used 10x, this would help me because it should show really bright bands because of the increase in concentration.<br />
<br />
Matt and
I had a discussion the reasons why I had not gotten successful results for all six
bacteria; we looked over my abstract and talked about maybe doing one of the
steps different. This step included setting my extractions aside for an entire
hour, then placing them in an 80 degree water bath for 2 minutes, and then continuing with the gel. I made sure to use the 1x sybr green dye that I usually use, and noticed how very light the color of the bands seemed compared to the first two results. <br />
<br />
Figure 1 and 2 show the difference the concentration of agarose makes ( you can see the DNA better in Figure 2). Figure 3 shows the DNA in 1% agarose gel, but shows only a few bacteria. <br />
<span style="mso-spacerun: yes;"> <table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; height: 134px; margin-right: 1em; text-align: left; width: 101px;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhP0RBvIWB3NjH85HRZ6-b9VEO_IPN4OoSM-Nsj77wFuu2AbT4cD7qzQTMmf0Zw7DP2yRV0dznjBxZ2ai-cMIJgTw6_k-D06iQiex6rnxaN41eL4zrgfCPl7U4RIYdnVkRQCFvll631_GZX/s1600/UVP00097.JPG" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhP0RBvIWB3NjH85HRZ6-b9VEO_IPN4OoSM-Nsj77wFuu2AbT4cD7qzQTMmf0Zw7DP2yRV0dznjBxZ2ai-cMIJgTw6_k-D06iQiex6rnxaN41eL4zrgfCPl7U4RIYdnVkRQCFvll631_GZX/s1600/UVP00097.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1. <br />
.5% Agarose Gel<br />
Ruler<br />
<em>E. coli</em><br />
<em>S. aureus</em><br />
<em>B. subtilis</em><br />
<em>S. enterica</em><br />
<em>P. stuartii</em><br />
<em>S. marcescens</em><br />
</td></tr>
</tbody></table>
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; height: 144px; margin-left: 1em; text-align: right; width: 172px;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj8d7VuDUq-gwZ1-BqXoXdYnUKe5UuJb1U4p6FwdDzZZRAWbQ682zDm-6xTJujmYz-vBEL_4Ke7fNdL7siWitAlKN19uJkQhbO9XSPggInlzhJYWWbHwAqpEDdCfRGhNIKu9g3NYL1QA8jy/s1600/UVP00098.JPG" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj8d7VuDUq-gwZ1-BqXoXdYnUKe5UuJb1U4p6FwdDzZZRAWbQ682zDm-6xTJujmYz-vBEL_4Ke7fNdL7siWitAlKN19uJkQhbO9XSPggInlzhJYWWbHwAqpEDdCfRGhNIKu9g3NYL1QA8jy/s1600/UVP00098.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 2<br />
1% Agarose Gel<br />
Ruler<br />
<em>E. coli</em><br />
<em>S. aureus</em><br />
<em>B. subtilis</em><br />
<em>S. enterica</em><br />
<em>P. stuartii</em><br />
<em>S. marcescens</em> </td></tr>
</tbody></table>
<br />
<br />
<br />
<br />
</span><br />
<br />
<div class="separator" style="clear: both; text-align: center;">
</div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: 1em; margin-right: 1em; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgt7_og7SAikzjO8A392LbKwTelolJNxBORQGj5wkoR2EXWftRgM5DXO96MNfPkaHm3Oj8o6iMq1sbTOglyDyYtZEBZopRjl4StXfKfxWTEm19vxc4XwCOBR0asHrafQERDqnsIhyVNi5qY/s1600/UVP00099.JPG" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgt7_og7SAikzjO8A392LbKwTelolJNxBORQGj5wkoR2EXWftRgM5DXO96MNfPkaHm3Oj8o6iMq1sbTOglyDyYtZEBZopRjl4StXfKfxWTEm19vxc4XwCOBR0asHrafQERDqnsIhyVNi5qY/s1600/UVP00099.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 3<br />
1% Agarose Gel <br />
Ruler<br />
E. coli <br />
S. aureus<br />
B. subtilis<br />
S. enterica<br />
S. marcescens<br />
P. stuartii</td></tr>
</tbody></table>
cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-48733884488281704222013-03-20T00:00:00.001-07:002013-03-21T09:34:22.439-07:00Back at it! :)<br />
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;">After being away from the lab for so long I glad to be back!
I came in Monday to talk to my advisors about some internship opportunities
over the summer. They were able to advise me about the qualities that someone
looks for in an intern and gave some tips which cleared some questions up for
me. Besides their awesome advice I was able to get some cultures done so when I
would come in Tuesday I would have bacteria to work with. Since I am continuing
with my<span style="mso-spacerun: yes;"> </span>project my duty now is to test
every bacteria I have access to in the lab. This means I will be working with
new bacteria however I will continue to use the four main ones I have been
working on. I prepared seven bacteria and incubated them over night these seven
are:</span></div>
<ul>
<li><span style="font-family: Calibri;">Salmonella</span></li>
<li><span style="font-family: Calibri;">S. Epidermidis</span></li>
<li><span style="font-family: Calibri;">Serratia Marcescens</span></li>
<li><span style="font-family: Calibri;">Providencia Stuartii</span></li>
<li><span style="font-family: Calibri;">E. coli</span></li>
<li><span style="font-family: Calibri;">Bacillus subtilis</span></li>
<li><span style="font-family: Calibri;">S. aureus</span></li>
</ul>
<br />
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;">On Tuesday I was ready to try the Sigma and Epicentre
protocol on these seven bacteria however I learned that we ran out of the
reagents for each of the kits. I will have to wait until we order and receive
them. Instead I tried the Isolation and Purification of Total Genomic DNA
protocol and if you all recall this protocol was the least successful for my
extractions. I tried it with only E. coli because it is always good to start
fresh in an experiment and have an idea of what the outcome should be.<span style="mso-spacerun: yes;"> </span>When I tried it back in December I only got
DNA for E. coli, the other three bacteria were not visible after the
electrophoresis gel run. I would like to try this protocol on these bacteria
once more since I have been able to gain more experience in the lab and my
skills have grown. Maybe this time I would be able to get positive results for
DNA extraction.</span></div>
<br />
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;"><span style="mso-spacerun: yes;"> </span>Also Josh gave me a
new task to do after I do my DNA extractions instead of running a PCR I would
do a Restriction Endonuclease Digestion on the DNA. This procedure is used to
somehow cut this DNA sequence and using PCR we can reproduce some of the chunks
of sequence that would be missing. I still do not have a clear understanding of
this new part of my experiment but my homework now is to look more into it in
order to get great results! I will keep you all in touch with this new project!
</span></div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhBso2rQpKmLb0a2PelekSUm5CjUi9LVhwPVsYtmH1vzYkcdBg745JkZi8mR9XXPNGAEQ6uIT2rEPJsVp158SHsYM5cUnGjek4IyIZEF00v41krAd3IfMcyl4Hz0CY2ugw4s2Y76ftvfkdp/s1600/UVP00095.JPG" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhBso2rQpKmLb0a2PelekSUm5CjUi9LVhwPVsYtmH1vzYkcdBg745JkZi8mR9XXPNGAEQ6uIT2rEPJsVp158SHsYM5cUnGjek4IyIZEF00v41krAd3IfMcyl4Hz0CY2ugw4s2Y76ftvfkdp/s1600/UVP00095.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Electrophoresis Gel Results<br />
First lane Ruler<br />
Second lane <em>E.coli</em></td></tr>
</tbody></table>
cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-51557355032317186042013-03-19T09:17:00.000-07:002013-03-19T09:17:19.069-07:00ASU Conference
<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj1rGHu3oR_eqd2YPJNPWYvQj2mLuzWw7gWpn2_Yq5kyTD9fI6Z-GNoeVsphw2pgreTkyySHwF-dcSVRRzgcDE7MPT4R9XJ4imlWm-mc7aSSmFxiT2t5S6enI-BSLxx4PvT8tTB-yC2CEd2/s1600/tbmnCkfYGRnXFPYZgGKZOZQwdEwEXkgBYkHAmCTRONLGAPmTwzLwDT2dh2hM3DbxK.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj1rGHu3oR_eqd2YPJNPWYvQj2mLuzWw7gWpn2_Yq5kyTD9fI6Z-GNoeVsphw2pgreTkyySHwF-dcSVRRzgcDE7MPT4R9XJ4imlWm-mc7aSSmFxiT2t5S6enI-BSLxx4PvT8tTB-yC2CEd2/s1600/tbmnCkfYGRnXFPYZgGKZOZQwdEwEXkgBYkHAmCTRONLGAPmTwzLwDT2dh2hM3DbxK.jpg" height="133" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><a href="mailto:MGE@MSA">MGE@MSA</a> conference</td></tr>
</tbody></table>
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;">Hello everyone! I hope everyone enjoyed their spring break!
I know I was able to relax and clean a lot of mess I had been avoiding since
the semester started! Anyway before spring break I had to chance to present my
project at the MGE@MSA conference, and for being my first time attending
something like this I felt pretty proud of myself! I had my poster ready and I
felt ready to present it, even though I was super nervous and anxious about it.
Especially when I got there and saw how great everyone’s posters were. I was
impressed by the variety of research topics and the amount of students out
there that have the same interests as me. It was also amazing how all of these
students where from all over the world and they were excited about being here
in Arizona a place that to me is no big deal. </span></div>
<br />
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;"><span style="mso-spacerun: yes;"> </span>It was a long day
but I have to admit I felt important.<span style="mso-spacerun: yes;"> </span>I
thought it was pretty neat how they provided breakfast and lunch for us and at
the end of the day when we got our certificates; felt accomplished.<span style="mso-spacerun: yes;"> </span>I know lots of my peers have been mentioning
the amount of judges that passed around asking questions some had a few and
others had only one but to be honest I cannot tell who my judge was! I was on
the lookout for my judge but I was just not able to figure it out I know I had
more than 2 people come and ask me questions some of them even gave me a few
suggestions either way I was very professional and answered their questions to
the best of my abilities. Like I mentioned before for being the first time I
feel like I did a decent job. I enjoyed this event and I was glad to be
fortunate in participating in it, and I was able to learn so many things from
different people. </span></div>
<br />
<div class="MsoNormal" style="margin: 0in 0in 10pt;">
<span style="font-family: Calibri;">The speakers were all great in proving to us why going to
graduate school is so important. Giving us their own life experiences as
examples to how they have gained so much from this was interesting to listen
to. I got to hear how graduate school opens up doors to great career
opportunities that in the end result in the good life. </span></div>
cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-78300370954854890962013-02-28T09:09:00.000-08:002013-02-28T09:40:51.077-08:00The End<!--[if gte mso 9]><xml>
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<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">This week was a stressful but
successful week. I say this because I was able to finish my project for the ASU
conference which is only a week away! But I have to say it
was tricky trying to get all my information neatly organized. Plugging the
information was the easy part of this project, but making it
all fit took so long. Playing with the colors, font, font size,
and all the effects kept me entertained for a while. I was able to
finish this over the weekend and on Monday when I came in Matt nicely went over
it and gave me some feedback. I was able to change a few things to
make it better and even though I personally like the pink color I
originally chose that looked cute, I changed it to a lighter
pink because the one I had was too bright. Anyway, since my project is
finished and Matt has already gone over it I want to see if
all of you would be willing to give me some feedback on my
project. It is in our dropbox for all of you who want to check it
out. </span></div>
<div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">Since my project is done with I
think it is only fair to share with you what my conclusions were. Let me catch
you all up with what my purpose for this project was, summarize all my results
and inform you what my conclusion is. </span></div>
<div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in;">
<span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">For starters the purpose for my
project was to test a variety of extraction kits and protocols that would
determine the most efficient method for running PCR. </span></div>
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<br /></div>
<div class="MsoNormal" style="line-height: normal; margin-bottom: 0.0001pt;">
<span style="font-family: "Times New Roman","serif"; font-size: 12pt;">I tested three different
extraction kits;</span></div>
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<li class="MsoNormal" style="line-height: normal; mso-list: l2 level1 lfo1; mso-margin-bottom-alt: auto; mso-margin-top-alt: auto; tab-stops: list .5in;"><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">Epicentre: QuickExtract Bacteria DNA Extraction, </span></li>
<li class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-list: l2 level1 lfo1; tab-stops: list .5in;"><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">Protocol Isolation and Purification of total genomic
DNA</span></li>
<li class="MsoNormal" style="line-height: normal; margin-bottom: 0.0001pt;"><span style="font-family: "Times New Roman","serif"; font-size: 12pt;">Sigma: GenElute Bacteria Genomic DNA Kit</span></li>
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<br /></div>
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<span style="font-family: "Times New Roman","serif"; font-size: 12pt;">I tested different bacteria with
each kit;</span></div>
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<li class="MsoNormal" style="line-height: normal; mso-list: l0 level1 lfo2; mso-margin-bottom-alt: auto; mso-margin-top-alt: auto; tab-stops: list .5in;"><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">AgroBacterium (gram-negative)</span></li>
<li class="MsoNormal" style="line-height: normal; mso-list: l0 level1 lfo2; mso-margin-bottom-alt: auto; mso-margin-top-alt: auto; tab-stops: list .5in;"><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">Bacillus subtilis (gram-positive)</span></li>
<li class="MsoNormal" style="line-height: normal; mso-list: l0 level1 lfo2; mso-margin-bottom-alt: auto; mso-margin-top-alt: auto; tab-stops: list .5in;"><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">Escherichia coli
(gram-negative) </span></li>
<li class="MsoNormal" style="line-height: normal; mso-list: l0 level1 lfo2; mso-margin-bottom-alt: auto; mso-margin-top-alt: auto; tab-stops: list .5in;"><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">Salmonella (gram-negative)
</span></li>
<li class="MsoNormal" style="line-height: normal; mso-list: l0 level1 lfo2; mso-margin-bottom-alt: auto; mso-margin-top-alt: auto; tab-stops: list .5in;"><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">Staphylococcus aureus (gram-positive)</span></li>
<li class="MsoNormal" style="line-height: normal; mso-list: l0 level1 lfo2; mso-margin-bottom-alt: auto; mso-margin-top-alt: auto; tab-stops: list .5in;"><span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; mso-fareast-font-family: "Times New Roman";">Staphylococcus epidermidis (gram-positive)</span></li>
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<b><span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Bacteria</span></b></div>
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<b><span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Epicentre</span></b></div>
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<b><span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Sigma Extractions</span></b></div>
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<b><span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Isolation and Purification of genomic DNA
Extractions</span></b></div>
</td>
<td style="background: #FF5698; height: 47.8pt; padding: 0in 5.4pt 0in 5.4pt; width: 44.4pt;" width="59"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<b><span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">PCR Results</span></b></div>
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<td style="background: #FFD7E9; height: 21.5pt; padding: 0in 5.4pt 0in 5.4pt; width: 80.1pt;" width="107"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">AgroBacterium tumefaciens</span></div>
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<td style="background: #FFD7E9; height: 21.5pt; padding: 0in 5.4pt 0in 5.4pt; width: .75in;" width="72"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Positive </span></div>
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<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Positive</span></div>
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<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Negative</span></div>
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<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Positive</span></div>
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<td style="background: #FFECF4; height: 13.7pt; padding: 0in 5.4pt 0in 5.4pt; width: 80.1pt;" width="107"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Bacillus subtilis</span></div>
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<td style="background: #FFECF4; height: 13.7pt; padding: 0in 5.4pt 0in 5.4pt; width: .75in;" width="72"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Positive</span></div>
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<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Negative</span></div>
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<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Positive</span></div>
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<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Escherichi coli</span></div>
</td>
<td style="background: #FFD7E9; height: 20.3pt; padding: 0in 5.4pt 0in 5.4pt; width: .75in;" width="72"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Positive</span></div>
</td>
<td style="background: #FFD7E9; height: 20.3pt; padding: 0in 5.4pt 0in 5.4pt; width: 62.05pt;" width="83"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Positive</span></div>
</td>
<td style="background: #FFD7E9; height: 20.3pt; padding: 0in 5.4pt 0in 5.4pt; width: 67.3pt;" width="90"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Positive</span></div>
</td>
<td colspan="2" style="background: #FFD7E9; height: 20.3pt; padding: 0in 5.4pt 0in 5.4pt; width: 50.95pt;" width="68"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Positive</span></div>
</td>
</tr>
<tr style="height: 11.95pt; mso-yfti-irow: 4;">
<td style="background: #FFECF4; height: 11.95pt; padding: 0in 5.4pt 0in 5.4pt; width: 80.1pt;" width="107"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Salmonella</span></div>
</td>
<td style="background: #FFECF4; height: 11.95pt; padding: 0in 5.4pt 0in 5.4pt; width: .75in;" width="72"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Positive</span></div>
</td>
<td style="background: #FFECF4; height: 11.95pt; padding: 0in 5.4pt 0in 5.4pt; width: 62.05pt;" width="83"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Negative</span></div>
</td>
<td style="background: #FFECF4; height: 11.95pt; padding: 0in 5.4pt 0in 5.4pt; width: 67.3pt;" width="90"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Negative</span></div>
</td>
<td colspan="2" style="background: #FFECF4; height: 11.95pt; padding: 0in 5.4pt 0in 5.4pt; width: 50.95pt;" width="68"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Negative</span></div>
</td>
</tr>
<tr style="height: 19.75pt; mso-yfti-irow: 5;">
<td style="background: #FFD7E9; height: 19.75pt; padding: 0in 5.4pt 0in 5.4pt; width: 80.1pt;" width="107"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Staphylococcus aureus</span></div>
</td>
<td style="background: #FFD7E9; height: 19.75pt; padding: 0in 5.4pt 0in 5.4pt; width: .75in;" width="72"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Positive</span></div>
</td>
<td style="background: #FFD7E9; height: 19.75pt; padding: 0in 5.4pt 0in 5.4pt; width: 62.05pt;" width="83"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Negative</span></div>
</td>
<td style="background: #FFD7E9; height: 19.75pt; padding: 0in 5.4pt 0in 5.4pt; width: 67.3pt;" width="90"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Negative</span></div>
</td>
<td colspan="2" style="background: #FFD7E9; height: 19.75pt; padding: 0in 5.4pt 0in 5.4pt; width: 50.95pt;" width="68"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Positive</span></div>
</td>
</tr>
<tr style="height: 18.55pt; mso-yfti-irow: 6; mso-yfti-lastrow: yes;">
<td style="background: #FFECF4; height: 18.55pt; padding: 0in 5.4pt 0in 5.4pt; width: 80.1pt;" width="107"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Staphylococcus epidermidis </span></div>
</td>
<td style="background: #FFECF4; height: 18.55pt; padding: 0in 5.4pt 0in 5.4pt; width: .75in;" width="72"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Positive</span></div>
</td>
<td style="background: #FFECF4; height: 18.55pt; padding: 0in 5.4pt 0in 5.4pt; width: 62.05pt;" width="83"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Negative</span></div>
</td>
<td style="background: #FFECF4; height: 18.55pt; padding: 0in 5.4pt 0in 5.4pt; width: 67.3pt;" width="90"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Negative</span></div>
</td>
<td colspan="2" style="background: #FFECF4; height: 18.55pt; padding: 0in 5.4pt 0in 5.4pt; width: 50.95pt;" width="68"><div class="MsoNormal" style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0in; mso-element-anchor-horizontal: page; mso-element-anchor-vertical: paragraph; mso-element-frame-hspace: 9.0pt; mso-element-left: 263.9pt; mso-element-top: 22.9pt; mso-element-wrap: around; mso-element: frame; mso-height-rule: exactly;">
<span style="color: black; font-family: "Times New Roman","serif"; font-size: 10.0pt; mso-fareast-font-family: "Times New Roman";">Negative</span></div>
</td>
</tr>
<tr height="0">
<td style="border: none;" width="107"><br /></td>
<td style="border: none;" width="72"><br /></td>
<td style="border: none;" width="83"><br /></td>
<td style="border: none;" width="90"><br /></td>
<td style="border: none;" width="9"><br /></td>
<td style="border: none;" width="59"><br /></td>
</tr>
</tbody></table>
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<span style="font-family: "Calibri","sans-serif"; font-size: 11pt; line-height: 115%;"> The table you see is the same table I included
in my poster, this table shows which kits and protocols worked for each
bacteria and the last column shows which bacteria had positive results after
running PCR on them. If you can see the protocol that had the least amount of
successful extractions was the Isolation and Purification of genomic DNA, while
Epicentre had the most successful extractions and the Sigma worked for the gram-negative bacteria.</span><br />
<div style="direction: ltr; language: en-US; margin-bottom: 0pt; margin-top: 0pt; text-align: left; unicode-bidi: embed; vertical-align: baseline;">
</div>
<ul>
</ul>
cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-42777735218835402252013-02-21T14:16:00.002-08:002013-02-28T09:47:23.849-08:00Project TimeHello everyone, I hope you are all doing great! I know as
the days go by we all just get busier; I am pretty sure now more than ever
since our conference is only two weeks away. How exciting, I know I am both
excited and anxious about it. <o:p></o:p><br />
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
This is exactly what I have been focusing on this week. I
must say doing the experiments and writing observations down as well as trying
to figure out what went wrong or modify something for better results is
interesting and fun. On the other hand it is completely different from gathering
all this information and writing everything down in a formal way. This can be a
bit challenging for me, but not impossible. I am now in the process of
finishing my project and I am glad to say I am only two sections away from
being done. Yay!<o:p></o:p></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
On another note I have not shared with all of you the last
few PCR results that I got for a variety of primers and extractions. After I
got positive results for using primers 3 and 4 from the QuickExtract Bacterial
DNA Extraction Kit I tried primer 7 and my results were great! Gave me a boost
in self-esteem when I thought it would never work for me. I also tried a
different protocol the Sigma: GenElute Bacterial Genomic DNA Kit to extract DNA
from gram-negative bacteria, these happened to be E. coli and Agrobacterium.
When I ran the electrophoresis gel I had great results, I was successful in the
extraction for both bacteria, however Agrobacterium DNA was way more
concentrated than E. coli and I can say this by noticing how bright Agrobacterium’s’
DNA looked compare to E. coli’s’. I must say this protocol took longer than the
QuickExtract Bacterial DNA Extraction protocol but I was extremely content with
the results. The next step after getting positive results was running the DNA
samples in the Thermal Cycler for a PCR. For these two samples I used four
different primers, these primers are 3, 4, 6, and 7. I was once again satisfied
with the results because the polymerase chain reaction worked for both bacteria.
I must share how after running the DNA samples in the thermal cycler my results
showed more concentration for the E. Coli DNA and less for the Agrobacterium
DNA. I am now on the hunt to figuring out why</div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgbI6aUMnxte_yZ4AHVmcokKLAnRVNH-pnStTAfn_xgQv1KMT4nBkFtZj9mCXCKFZ_OhCuCHVvW15-VOxsFOlaXdQ8IEinFS87SvtkfuampaQ_1er1nE_W-6Oay_YkCyCGLBOK3K1905LHd/s1600/mail.google.com.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgbI6aUMnxte_yZ4AHVmcokKLAnRVNH-pnStTAfn_xgQv1KMT4nBkFtZj9mCXCKFZ_OhCuCHVvW15-VOxsFOlaXdQ8IEinFS87SvtkfuampaQ_1er1nE_W-6Oay_YkCyCGLBOK3K1905LHd/s1600/mail.google.com.jpg" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">PCR Results for Primer 7</td><td class="tr-caption" style="text-align: center;"><br /></td></tr>
</tbody></table>
<div class="MsoNormal">
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;">Ruler, E. Coli, S. Aureus, Bacillus, AgroBacterium</td><td style="text-align: center;"><br /></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" style="text-align: center;"></td><td class="tr-caption" 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cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-76526742968668809242013-02-14T09:59:00.000-08:002013-02-14T10:05:43.269-08:00PCR Time ! <div class="separator" style="clear: both; text-align: center;">
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<span id="internal-source-marker_0.019317635621812712" style="background-color: transparent; color: black; font-family: Arial; font-size: 15px; font-style: normal; font-variant: normal; font-weight: normal; text-decoration: none; vertical-align: baseline;">Last week I worked on my extractions. I did about three extractions using the same protocol and the same bacteria. If you have been following up with my experiments you now know that the last extraction was definitely the best I have done and the results from the gel electrophoresis can prove it. Now that I have mastered the extraction part of my experiment running a PCR for each bacteria should be easy. </span><br />
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: 15px; font-style: normal; font-variant: normal; font-weight: normal; text-decoration: none; vertical-align: baseline;">Let me remind you that PCR stands for Polymerase Chain Reaction, and what this type of machine or technology tool does is it takes a piece of DNA and amplifies it by making several thousands to millions copies of a specific DNA sequence. This makes large and efficient amount of DNA that could be used for several tests. For example finding out suspects from a criminal act, finding out who a deceased body belongs to, or track down virus and diseases. However in my case I am just running this experiment to see how successful I can be. </span><br />
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: 15px; font-style: normal; font-variant: normal; font-weight: normal; text-decoration: none; vertical-align: baseline;">In order to even begin a PCR I must have the DNA of the bacteria extracted and ready to go, because I used E. Coli, Bacillus, S. Aureus, and Agrobacterium, I continued with the PCR protocol from these same extractions. The PCR works a little different from the gel electrophoresis because for one it takes six times longer! We add different reagents and components such as master mix, primers, DI water and of course DNA, the order in which we mix these four components and reagents together is important and when doing this we must keep them on ice. Primers are reagents that contain nucleic acid and are included in this mixture because this nucleic acid helps start the DNA synthesis. The master mix is a reagent that contains Taq polymerase, and this polymerase helps amplify short DNA segments. The addition of water helps dilute the concentrations of both reagents just mentioned and DNA has to be included. I ran two PCR the first one was with primers 4 and 5 with agrobacterium and E. Coli. The second PCR I tried was with primers 3, and 4 with E. Coli, S. Aureus, Bacillus, and Agrobacterium. To check if the PCR produced DNA I run a gel electrophoresis just like I run it for the extraction part. I was not successful with the first PCR and failed to capture a picture of it, but the second try was a lot better. </span><br />
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: 15px; font-style: normal; font-variant: normal; font-weight: normal; text-decoration: none; vertical-align: baseline;">PCR Steps</span><br />
<span style="background-color: transparent; color: black; font-family: Arial; font-size: 15px; font-style: normal; font-variant: normal; font-weight: normal; text-decoration: none; vertical-align: baseline;">1. Add 10 microliters of master mix</span><br />
<span style="background-color: transparent; color: black; font-family: Arial; font-size: 15px; font-style: normal; font-variant: normal; font-weight: normal; text-decoration: none; vertical-align: baseline;">2. Add 5 microliters of DI H2O</span><br />
<span style="background-color: transparent; color: black; font-family: Arial; font-size: 15px; font-style: normal; font-variant: normal; font-weight: normal; text-decoration: none; vertical-align: baseline;">3. Add 4 microliters of primer </span><br />
<span style="background-color: transparent; color: black; font-family: Arial; font-size: 15px; font-style: normal; font-variant: normal; font-weight: normal; text-decoration: none; vertical-align: baseline;">4. Add 1 microliters of DNA Sample</span></div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjzoMHhkmIYAYokqsd7DsAL_LhdAwC8xTK24Y9vyfld1MhvsQ4ZDEhqppFrBzxotlLw84k8Kr6A8sp6DgRk927NPJWY-D_aq5WjOlo7PVb_2SqolFE5gSrZGXzM6Dr4U4G5pXENLUdth99W/s1600/PCR+primer+4.JPG" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjzoMHhkmIYAYokqsd7DsAL_LhdAwC8xTK24Y9vyfld1MhvsQ4ZDEhqppFrBzxotlLw84k8Kr6A8sp6DgRk927NPJWY-D_aq5WjOlo7PVb_2SqolFE5gSrZGXzM6Dr4U4G5pXENLUdth99W/s1600/PCR+primer+4.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Primer4<br />
E. Coli, S. Aureus, Bacillus, Agrobacterium</td></tr>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh6sB4qC7sp_zz4gP-gEYYhIdTs16ScsZvTAbToTd1GujtFz3U3VQUG61CFH1ZmPt6KyVPDOVCfDDjXiE7VSL_pfQiXcG7SCjtjNeWdUAYbH_p3r1eq8VS4FDGX7HKrmDN1QOwqY0H-KviM/s1600/PCR+Primer+3.JPG" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh6sB4qC7sp_zz4gP-gEYYhIdTs16ScsZvTAbToTd1GujtFz3U3VQUG61CFH1ZmPt6KyVPDOVCfDDjXiE7VSL_pfQiXcG7SCjtjNeWdUAYbH_p3r1eq8VS4FDGX7HKrmDN1QOwqY0H-KviM/s1600/PCR+Primer+3.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Primer 3<br />
E. Coli, S. Aureus, Bacillus, Agrobacterium </td></tr>
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cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-31193478152147220212013-02-07T22:13:00.001-08:002013-02-07T22:13:27.911-08:00Third times the charm<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiaQkG73ldRPPzBQBTj-t76I67yvS3rVwOO5b88x3PgARvuHAaa2OxeyCiYamMtyP_3NJTQsx9O-HNjVOvccidC6DP6kTGyuzkKCO06N7dKkC2pvz8NVK3wprXbWQ9JQJ7QEmM0bX5He3KG/s1600/UVP00064.JPG" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"></a><br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgrcpgSWsPMNV3B3HmhJTUYXYO8khtsKqCNn56MGA87CpLkd6NYi7KUrCprK1eDz6dIHH2C9-N0KLmwKMlUKQ5nhr25eAED89qPpsgo5EaW6v3PKYIT59RuF5QLc7R54pUPCaB1ZwJJSR2j/s1600/UVP00064.JPG" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgrcpgSWsPMNV3B3HmhJTUYXYO8khtsKqCNn56MGA87CpLkd6NYi7KUrCprK1eDz6dIHH2C9-N0KLmwKMlUKQ5nhr25eAED89qPpsgo5EaW6v3PKYIT59RuF5QLc7R54pUPCaB1ZwJJSR2j/s1600/UVP00064.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1 <br />
Ruler E. Coli S Aureus, Bacillus Agrobacterium<br />
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Last week I worked on my DNA extractions. The bacteria I worked on were E. Coli, S. Aureus, Bacillus, and Agrobacterium. To test if my extractions were successful I ran a gel for each. I have some pictures of what I see after running the gel. I allowed the gel to run for 35 minutes at 120 volts, when that was done I placed it under UV light, and that light allows us to see if DNA is present or not. The reason we check if DNA is present this way is because to each sample I add two different dyes if any of you recall, one of them (orange dye) gives the DNA bonds higher resolution in the agarose gel while the syber green stains the nucleic acid bonds from the DNA allowing it to absorb blue light (UV light) and emits a green color. When I place the gel under this UV light I should be able to see green light for each of my samples showing the existence of DNA including the ruler. For example in figure 1 (my first extraction), you can only see about three bacteria not including the ruler. The ruler has about 5 bright dots in its row. You can only see a big dot for E. Coli, a small dot for Bacillus, and just a line for Agrobacterium. This extraction worked for those three bacteria but failed for the S. Aureus, but none of the samples actually ran all the way down like the ruler did. This gel is 1% agarose a normal percentage to use for a gel, but in this case this gel was too concentrated and for this reason the samples did not run all the way down like they should have. If you notice the only bright bands you see at the other side of the rectangle is the rulers, not the bacteria's DNA. On figure 2 (my second extraction) though, you can see a bright light for all of them, even though you can barely see the S. Aureus band. This extraction worked for all four but even though the gel concentration was changed to .5%, the samples did not run all the way down. Figure 3 (my third extraction) though is the best extraction I have done so far. You can notice the difference in the brightness, and the length that each travelled ( almost to the ruler). By length I mean how far each sample ran to the bottom part of the rectangle. <br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiJM6gjdxPueqBcIITPD0_ilNTTcquJ-ukgDdSSnO9qnkzBavAS3lOtVjr1dW0ZSje5bjLcotA_z3vHQI4YwAxZLGu0ovzZztZmg_ZmfuIuUGfL1LDzZ6i9bubvRfKLM-cR5I-9FLg58Sbp/s1600/UVP00066.JPG" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiJM6gjdxPueqBcIITPD0_ilNTTcquJ-ukgDdSSnO9qnkzBavAS3lOtVjr1dW0ZSje5bjLcotA_z3vHQI4YwAxZLGu0ovzZztZmg_ZmfuIuUGfL1LDzZ6i9bubvRfKLM-cR5I-9FLg58Sbp/s1600/UVP00066.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Fgure 2<br />
Ruler E. Coli S Aureus, Bacillus Agrobacterium<br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjkkFIXkw_0nMmNCCB_mMPXhPQs3zybRGxHTPuOXenACBJr4k2a7b3vE4zpr6etmOmzXHwjPaxUwEyEU6F38xxDs6lTIU2blstZ9bhHxG211Oh6U4EuCky9DVe53BnUJpaYmFYWLx0JEty8/s1600/UVP00069.JPG" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjkkFIXkw_0nMmNCCB_mMPXhPQs3zybRGxHTPuOXenACBJr4k2a7b3vE4zpr6etmOmzXHwjPaxUwEyEU6F38xxDs6lTIU2blstZ9bhHxG211Oh6U4EuCky9DVe53BnUJpaYmFYWLx0JEty8/s1600/UVP00069.JPG" height="151" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 3<br />
Ruler E Coli S Aureus Agrobacterium Bacillus<br />
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cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com1tag:blogger.com,1999:blog-3762715904401374837.post-82301015984741212362013-01-30T08:46:00.000-08:002013-01-31T09:56:20.319-08:00QuickExtract Bacterial DNA Extraction Kit<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEihQUD20AATViJL8gDuSARhU909qAoBQguWS4U1pYTkaf_qvMr6ROD9WUWLrTNXEwmsslRPchDtzaWLHG7XwDXyHf7qxLy5H9hYrXcr5dQn_IJJWVTpchmk8_gp6-sfLlMh_gYgUHN0IzpA/s1600/PART_1359654039300.jpeg" imageanchor="1" style="clear: right; cssfloat: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEihQUD20AATViJL8gDuSARhU909qAoBQguWS4U1pYTkaf_qvMr6ROD9WUWLrTNXEwmsslRPchDtzaWLHG7XwDXyHf7qxLy5H9hYrXcr5dQn_IJJWVTpchmk8_gp6-sfLlMh_gYgUHN0IzpA/s1600/PART_1359654039300.jpeg" ea="true" height="150" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Equitment for DNA Extraction</td></tr>
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This week for my internship I had the chance to proceed with my DNA extraction experiment. Remember my goal for all of these experiments is to fine an efficient way to extract DNA from bacteria that will create clear PCR product.</div>
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Recall that last week I made cultures for four different bacteria, E.Coli, S. Aureus, Bacillus, and Agrobacterium. I continued with the extraction procedure the next day. A summary of my procedures for the DNA extraction are the following:</div>
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<li style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;"> Extract 500microliters of culture into a flat cap tube.</li>
<li style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">Centrifuged the bacteria at 1300rmp in a micro centrifuge for 3 minutes. (To pellet the cells)</li>
<li style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">Extract the liquid (leaving only the pellet of cells) and add .5ml of sterile water.</li>
<li style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">Recentrifuge at same speed as before for 3 minutes.</li>
<li style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">Remove and discard the supernatant, and add 100 microliters of QuickExtract Bacterial DNA Extraction Solution to cell pellet.</li>
<li style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">Add 1 microliter of Ready-Lyse Lysozyme Solution to each tube and mix.</li>
<li style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;"> Incubate at room temperature for 15 minutes. </li>
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When all these steps were done for the four tubes, they were ready for either PCR, or for running a gel. However because both of these steps require time they were incubated at 4 Celsius degrees, and left there over weekend. </div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhQ95Z9giaugcLUpmdjQIWWmenYtmO67EK3lG7P0T47jSPMIXeoCuifVruUXyArR43lsV62ikvbYbxYE0WXQCjHbS0eiqdku5ojTr6Mk44euYH3PQWNCbTBT_1b_gf9Gu4KtjqB9FhBWYzi/s1600/PART_1359653992718+-+Copy.jpeg" imageanchor="1" style="clear: left; cssfloat: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhQ95Z9giaugcLUpmdjQIWWmenYtmO67EK3lG7P0T47jSPMIXeoCuifVruUXyArR43lsV62ikvbYbxYE0WXQCjHbS0eiqdku5ojTr6Mk44euYH3PQWNCbTBT_1b_gf9Gu4KtjqB9FhBWYzi/s1600/PART_1359653992718+-+Copy.jpeg" ea="true" height="150" width="200" /></a></div>
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<tr><td class="tr-caption" style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none; text-align: center;">Molecular Weight Ruler</td></tr>
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On Tuesday, I ran an agarose <span lang="EN" style="mso-ansi-language: EN; mso-bidi-font-weight: bold;">gel electrophoresis (method used to separate DNA based on its size). Syber Green is a blue/green cyanine dye used to stain nucleic acid bonds to DNA (Absorbs blue light and emits green). Orange loading dye gives bonds higher resolution in the agarose gel. Both dyes are used for this procedure in addition a separate ruler to compare the results of the DNA. The electrical field that is applied to this apparatus is set to 120 volts, the entire box where the agarose and DNA are is completely covered and it runs for 35 minutes. </span> <br />
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<tr><td class="tr-caption" style="text-align: center;">Syber Green and Orange Dye</td></tr>
</tbody></table>
<div style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">
<br /></div>
<div style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">
<br /></div>
<div style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">
<span lang="EN"><strong>Flat Cap Tubes for </strong></span><span lang="EN"><strong>Agarose Gel Contain:<span style="mso-spacerun: yes;"> </span></strong></span></div>
<ul>
<li><div style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">
<span lang="EN" style="mso-ansi-language: EN; mso-bidi-font-weight: bold;">8 microliters of DNA</span></div>
</li>
<span lang="EN" style="mso-ansi-language: EN; mso-bidi-font-weight: bold;"><span style="mso-spacerun: yes;">
<li><div style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">
2 microliters of Orange Dye<span style="mso-spacerun: yes;"> </span> </div>
</li>
<li></li>
</span></span><span lang="EN" style="mso-ansi-language: EN; mso-bidi-font-weight: bold;">1 microliters of Syber Green</span></ul>
<br />
<div style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">
<strong><span lang="EN"> Ruler Contains:</span></strong></div>
<ul>
<li><div style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">
9 microliters of Molecular Weight ruler</div>
</li>
<li style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">1 microliter of Syber Green</li>
</ul>
<div>
</div>
<div style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">
<span lang="EN">After running agarose gel electrophoresis I used the help of the UV light to see if the DNA extraction was done correctly for all except the S. Aureus, and neither of the bacterias travelled within the agarose. When I tried it a second time the results were almost the same, (a bit clearer) but still not one travelled throughout the agarose, only the ruler. </span></div>
<div style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">
<br /></div>
<div style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none;">
<br /></div>
<br />
<div class="separator" style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none; clear: both; text-align: center;">
</div>
<div class="separator" style="border-bottom: medium none; border-left: medium none; border-right: medium none; border-top: medium none; clear: both; text-align: center;">
</div>
cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-33059155472525817072013-01-24T09:59:00.000-08:002013-01-27T20:18:16.818-08:00Fresh Start<!--[if gte mso 9]><xml>
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After a long break, I can honestly say I am glad to be back.
I am ready for what is my last semester at Phoenix College, and excited to
start my internship with Matt and Josh.</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
Today was my first day back and because I applied for the
MGE Conference for ASU my advisers and I thought it would be a good idea to
redo all of my experiments that were done last semester. For those of you who
have no idea what my project consist of it has to do with finding the most
efficient method for running a PCR. Efficiency is determined by several
variables, including cost, quantity and quality of DNA, time required for
extraction, and which method produced clear PCR product. In order to be able to
run a PCR I first have to successfully extract DNA from bacteria. With that my
job today was to make cultures for the several different bacteria that I will
be using in both the Quick Extract Bacterial DNA Extraction Kit Protocol and
the Isolation and Purification of Total Genomic DNA Protocol.</div>
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj2_kgVzqhNOQMNCiOG6tMM9hIJtrZI94IEhgJnOo3r-dHqPhhmSqDHkbZjsrtUgwMlRqzsw9KMo-xu-iR75Kgg05JYBNW7KsBfs1OlBC-nbNnZi394BE1JDzXJu-1SKEyplWpE_m19xFyk/s1600/PART_1359046023621.jpeg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj2_kgVzqhNOQMNCiOG6tMM9hIJtrZI94IEhgJnOo3r-dHqPhhmSqDHkbZjsrtUgwMlRqzsw9KMo-xu-iR75Kgg05JYBNW7KsBfs1OlBC-nbNnZi394BE1JDzXJu-1SKEyplWpE_m19xFyk/s1600/PART_1359046023621.jpeg" height="150" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Rack where Bacteria is stored </b></td><td class="tr-caption" style="text-align: center;"><br /></td></tr>
</tbody></table>
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj5FMhf6M_RmOoSqH9is1mOCK5mRf9X0A_Cdr-ypgu2Wx6wvbDTUOIaemLcUcDGqr7Uw4xUHcrUboQDF2-IKry2M0geUO_0boR1NtFgmdOc4i7SMa8Z34nz2McSNjF6D6yrFj3wAYxnkmj_/s1600/PART_1359045997257.jpeg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj5FMhf6M_RmOoSqH9is1mOCK5mRf9X0A_Cdr-ypgu2Wx6wvbDTUOIaemLcUcDGqr7Uw4xUHcrUboQDF2-IKry2M0geUO_0boR1NtFgmdOc4i7SMa8Z34nz2McSNjF6D6yrFj3wAYxnkmj_/s1600/PART_1359045997257.jpeg" height="150" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><b>Rack with TSB tubes</b></td></tr>
</tbody></table>
<div style="text-align: center;">
</div>
<div class="MsoNormal" style="text-align: left;">
<br /></div>
<br />
<br />
<br />
<b><u></u></b><br />
<b><u></u></b><br />
<b><u></u></b><br />
<b><u></u></b><br />
<b><u></u></b><br />
<b><u></u></b><br />
<b><u></u></b><br />
<b><u>Procedures
</u></b><br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgShCPP6B3EdWD46pL7LUcCwEFqrTdBFcoMEzibMkQfCY5J6yxqoHC3ZIljuGv40zJI7tIQum_O8VrBaBlHSg0NWVOrBZVQwCIRdKp9Y-kGWoz5bHULyGm8Pb81cytGKsK1AKqrv5ZzKnAs/s1600/PART_1359046048566.jpeg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgShCPP6B3EdWD46pL7LUcCwEFqrTdBFcoMEzibMkQfCY5J6yxqoHC3ZIljuGv40zJI7tIQum_O8VrBaBlHSg0NWVOrBZVQwCIRdKp9Y-kGWoz5bHULyGm8Pb81cytGKsK1AKqrv5ZzKnAs/s1600/PART_1359046048566.jpeg" height="150" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: left;"><div style="text-align: center;">
<b>My four cultures</b></div>
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td class="tr-caption" style="text-align: left;"><b>E.Coli</b></td><td class="tr-caption" style="text-align: left;"><b><br /></b></td><td class="tr-caption" style="text-align: left;"><b> Bacillus Agrobacterium S. Aureus</b></td><td class="tr-caption" style="text-align: left;"><b><br /></b></td><td class="tr-caption" style="text-align: left;"><b> </b></td></tr>
</tbody></table>
</td><td class="tr-caption" style="text-align: left;"><br /></td><td class="tr-caption" style="text-align: left;"><br /></td><td class="tr-caption" style="text-align: left;"><br /></td><td class="tr-caption" style="text-align: left;"><br /></td><td class="tr-caption" style="text-align: left;"><br /></td><td class="tr-caption" style="text-align: left;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td><td class="tr-caption" style="text-align: center;"><br /></td></tr>
</tbody></table>
<div class="MsoNormal">
Before anything was done, I made sure to label each TSB tube
to its corresponding bacteria. The 4 TSB tubes are needed because I have to add a few drops of
bacteria in each, helping the bacteria grow. However before adding these drops
into the TSB tubes each bacteria was gently pipetted allowing the bacteria to
mix, we do this to get rid of the bacteria clumps that are located at the
bottom of the tubes. After mixing it well two drops of each bacteria was added
to its corresponding TSB tube. These TSB tubes were all covered and incubated.
They have to be left in the incubator for a whole night to allow the bacteria
to grow in order to use them for the DNA extraction experiments.</div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="text-align: left;">
<u><b>Materials</b></u><span style="mso-spacerun: yes;"><u><b> </b></u> </span><span style="mso-spacerun: yes;"> </span></div>
<div class="MsoNormal" style="text-align: left;">
<span style="mso-spacerun: yes;"></span><br /></div>
<div class="MsoNormal" style="text-align: left;">
E. Coli<span style="mso-spacerun: yes;"> </span></div>
<div class="MsoNormal" style="text-align: left;">
<span style="mso-spacerun: yes;"></span>S. Aureus</div>
<div class="MsoNormal" style="text-align: left;">
Bacillus</div>
<div class="MsoNormal" style="text-align: left;">
Agrobacterium</div>
<div class="MsoNormal" style="text-align: left;">
4 TSB Tubes </div>
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td class="tr-caption" style="text-align: left;"><br /></td><td class="tr-caption" style="text-align: left;"><br /></td><td class="tr-caption" style="text-align: left;"><br /></td><td class="tr-caption" style="text-align: left;"><br /></td><td class="tr-caption" style="text-align: left;"><br /></td></tr>
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<![endif]--><br />cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com2tag:blogger.com,1999:blog-3762715904401374837.post-67093577449898360162012-12-16T21:03:00.001-08:002012-12-16T23:06:19.699-08:00Plan BIn the previous blog I talked about the main goal of my project; to find the most beneficial way to extract DNA from bacteria that will run in two different techniques; gel extraction and in the PCR (polymerase chain reaction). In the last experiment I was successful in running the gel extraction with three bacterias; Salmonella, E. Coli, and S. Aurues, but not successful in the PCR technique. <br />
The next day after finding out that the PCR did not work, it was time to test a different protocol. This protocol is called "Isolation and Purification of Total Genomic DNA from E Coli", an experiment that is longer compared to my first experiment.<br />
The procedures for this experiment began just like the first experiment. I had to grow bacteria over night for 24 hours in a TSB tube, but since I had my bacterias already growing from the first experiment I went ahead and used the same ones (E. Coli, S. Aureus, S. Epidermidis, and Salmonella). My S. Epidermidis tube was spilled when transferring so I used a different TSB tube that was grown a week earlier. I had to do some centrifuging at high speeds for intervals of 2-3 minutes. As well as some incubating at 37-80 degrees Celcius for 30 minutes and 5 minutes. Lysis solution was added to disrupt membranes and denature proteins. RNase solution was added to degrade RNA into small fragments or ribonucleotides. Protein Precipitation Solution to add insoluble material for the centrifuging step. Isopropanol was added to the solution, also 70% ethanol, and lastly the DNA Rehydration Solution (TRIS EDTA Buffer). In the end of all these procedures the extractions made were ready to run in either techniques. The first one I did was the gel extraction, the gel was made out of 100 milliliters of TAE and 1 gram of molecular Biology Agarose. When analyzing the gel under uv light, I came to the conclusion that this extraction only worked for the E. Coli and not for the rest. Once again a PCR was not done because I had no DNA from the extraction to run. Here are some pictures enjoy ! <br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjTXxD9KXlGKdBVJCggeazf2JKX9ueWeCke2yLQhyphenhyphen3-Gj8XWeR3xMln0T_Q7FC3vC_U9Rn9EYzQNWOVmgSANWHP1mrSo3WWr7hWish-QP4vp6bbE-YZqI1fh_R3DNF3k5ihZBzU4Lwkgq-5/s1600/PART_1355726685666.jpeg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjTXxD9KXlGKdBVJCggeazf2JKX9ueWeCke2yLQhyphenhyphen3-Gj8XWeR3xMln0T_Q7FC3vC_U9Rn9EYzQNWOVmgSANWHP1mrSo3WWr7hWish-QP4vp6bbE-YZqI1fh_R3DNF3k5ihZBzU4Lwkgq-5/s1600/PART_1355726685666.jpeg" height="200" width="150" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Closest to 80 degree Celsius <br />
the incubator reached. </td></tr>
</tbody></table>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi-WvbB_eMi9geU9Hp0hda65uuBey-LUO9Cexx8x-oCX5iz92Ery6e4yGvh-JBTbCexibiluWuR5TbyEnMNgyVNa9ZlOdpc-l9_0JBT9EPLitbfsriclVWnXvLtJpjxtbqD2AemguHhqtIb/s1600/IMG_20121211_135236.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi-WvbB_eMi9geU9Hp0hda65uuBey-LUO9Cexx8x-oCX5iz92Ery6e4yGvh-JBTbCexibiluWuR5TbyEnMNgyVNa9ZlOdpc-l9_0JBT9EPLitbfsriclVWnXvLtJpjxtbqD2AemguHhqtIb/s1600/IMG_20121211_135236.jpg" height="200" width="150" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Gel Extraction Apparatus</td></tr>
</tbody></table>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjsB4P6TBbcQKpsOj6ssTYEiPzIX-9o17e6xsBpuXMEegHnB3oArndiA6XsP1s5GwzFKSYmeVUfXWI1lY-INdOkSQOALqhOWgrotZDryD15YZ8xGhIA1EJve3eSDajdKONOQ89vb0iw0wAs/s1600/IMG_20121211_135328.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjsB4P6TBbcQKpsOj6ssTYEiPzIX-9o17e6xsBpuXMEegHnB3oArndiA6XsP1s5GwzFKSYmeVUfXWI1lY-INdOkSQOALqhOWgrotZDryD15YZ8xGhIA1EJve3eSDajdKONOQ89vb0iw0wAs/s1600/IMG_20121211_135328.jpg" height="200" width="150" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">TAE Solution </td></tr>
</tbody></table>
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cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-75276388496232252382012-12-14T19:29:00.000-08:002012-12-14T19:38:34.439-08:00Lets do this !<br />
After a week of deciding what my project would consist of, DNA Extraction came to the rescue. I was introduced to the Extraction of Bacterial DNA from Gram-Positive and Gram-Negative species experiment. The goal of this project is to find the best method to extract DNA from various bacteria that will efficiently and successfully run in the PCR (polymerase chain reaction). The PCR is a machine that is used to amplify either a single or a few copies of DNA resulting in the duplication of several, to millions of copies of a specific DNA section. However to actually run a PCR we have to first extract the DNA and prove that there is DNA present to run it, most of my time was dedicated to this first step of the project. In order to find out if DNA is really gained from the bacteria extraction a gel extraction technique was used to prove it. The gel extraction is a different type of technique that isolates the DNA from an agarose gel, and with the help of uv light we know if DNA is present from the extraction done. <br />
<br />
The first protocol I tested was short and simple and glad to say successful. Just a few steps were required as opposed to the second protocol which was more complex. For both, I made a culture for four different bacterias: E. Coli, S. Aureus, S. Epidermidis, and Salmonella. I placed a drop of each into their own TSB (tryptic soy broth) tubes and allowed the bacteria to grow for 24 hours. The next day I noticed that the S. Epidermidis did not have any bacteria growth, compared to the other three. I had to centrifuge each of them for 3 minutes under 5000 rmp, then extracted the TBS. I washed the bacterial cell pellet with.5ml sterile water following up with 3 minutes in the centrifuge. After the pellet was washed I extracted the supernatant and added 100microliters of QuickExtract Bacterial DNA Extraction Solution. With that I added 1 microliters of Ready-Lyse Lysozyme Solution to each of the tubes. After a few minutes of incubation at room temperature the tubes were ready for the gel extraction and the PCR.<br />
<br />
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<tr><td style="text-align: center;"> <a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjfcc3DFSjLSMqW-Jw9oZ6RhxvzfAc4FUvGsYs6V5GPQuaOBFl3m1aL5xq7IkZTFj2AdRRXLc5H92RkWAg6pN-ANvQer4pwNzwGu6IueK_IHEm25YyQtWsxkElGXkE50zyCCiCFnEVgiq-R/s1600/PART_1355540333314.jpeg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjfcc3DFSjLSMqW-Jw9oZ6RhxvzfAc4FUvGsYs6V5GPQuaOBFl3m1aL5xq7IkZTFj2AdRRXLc5H92RkWAg6pN-ANvQer4pwNzwGu6IueK_IHEm25YyQtWsxkElGXkE50zyCCiCFnEVgiq-R/s1600/PART_1355540333314.jpeg" height="150" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Test tubes <br />
Top Row Ready for Gel Extraction<br />
Bottom Row Samples from TSB culture<br />
<br />
</td></tr>
</tbody></table>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiw5jikbzzB2Eqp-AxKkjDtFNaMlqKMtHLd_tPTxCucg1Go1qu_A_lUSBgVwgYZ7GAmj4DEqEy2D-1Azm-bva-LeYXwarwJmEuGjsmUp7g1rrPT63KcryM1DQzJ4_DzsOInkL0d2HmhY4xC/s1600/PART_1355540247673.jpeg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img alt="jajf" border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiw5jikbzzB2Eqp-AxKkjDtFNaMlqKMtHLd_tPTxCucg1Go1qu_A_lUSBgVwgYZ7GAmj4DEqEy2D-1Azm-bva-LeYXwarwJmEuGjsmUp7g1rrPT63KcryM1DQzJ4_DzsOInkL0d2HmhY4xC/s1600/PART_1355540247673.jpeg" height="150" title="" width="200" /></a> </td></tr>
<tr><td class="tr-caption" style="text-align: center;">Left = QuickExtract Bacterial DNA Extraction Solution<br />
Right = Ready-Lyse Lysozyme Solution <br />
<br />
<br />
</td></tr>
</tbody></table>
<br />
<br />
My results for the gel extraction were great ! Except for the S. Epidermidis, because just like I suspected the DNA extraction was not successful. You can see this by the really dark square next to the E.Coli. That same day left the PCR running with the salmonella but unfortunately the next day I found out that the PCR test did not work. This conclusion has left me with the second protocol to test. I will soon have results of which protocol gave the best results, and which one is able to continue with the PCR. <br />
<div class="separator" style="clear: both; text-align: center;">
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; height: 227px; margin-right: 1em; text-align: left; width: 92px;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiYkUGk96S2-IIxIiaIfpqmbHgkfv-UX3hqLhU6oIRUVsSTtTa9ph1RBMPSoUg8h69DFnzM3YsKbsOv0RUI9I8PH_9elTZnNMXF-Z9jw5Fw3oJazcWaQr0b53iL_q4dCOr3hbD4P-OBcBXF/s1600/image01.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiYkUGk96S2-IIxIiaIfpqmbHgkfv-UX3hqLhU6oIRUVsSTtTa9ph1RBMPSoUg8h69DFnzM3YsKbsOv0RUI9I8PH_9elTZnNMXF-Z9jw5Fw3oJazcWaQr0b53iL_q4dCOr3hbD4P-OBcBXF/s1600/image01.jpg" height="150" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Gel Extraction under UV Light <br />
<div align="left">
First Row Ruler</div>
<div align="left">
Second Bright Row E.Coli</div>
<div align="left">
Third Dark Row S. Epidermidis</div>
<div align="left">
Fourth Medium Row S. Aureus</div>
<div align="left">
Last Bright Row Salmonella</div>
</td></tr>
</tbody></table>
cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-52797996270106518932012-12-06T08:29:00.001-08:002012-12-06T08:51:28.109-08:00Strokes: Therapy, Drugs, & more <div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiymcwdW6T5RxjVULHs3FvsMlJmxj7BY8LRzY09djqlSRYEBmaoUsfYrrpt0gYdGGTT-MsXPjpcwegufKeWh5hiJX-M4fGb-BxJKxaLUuMS9IMgq7pt7AdHlJytyktSsrQE0DXxSci-fwYr/s1600/brain-components-diagram.gif" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiymcwdW6T5RxjVULHs3FvsMlJmxj7BY8LRzY09djqlSRYEBmaoUsfYrrpt0gYdGGTT-MsXPjpcwegufKeWh5hiJX-M4fGb-BxJKxaLUuMS9IMgq7pt7AdHlJytyktSsrQE0DXxSci-fwYr/s1600/brain-components-diagram.gif" height="160" width="200" /></a></div>
<br />
Restorative Neurology “Drugs and Recovery Following Stroke”<br />
<br />
This past week I have dedicated most of my time to an article I found that's main focus was the recovery process of a stroke. The main topic for this journal dealt with four main points, the biological and environmental factors of a stroke, the responses to the injury, neuronal rearrangements, and the adaptive responses. <br />
<br />
Thanks to laboratory work, these four factors can now be understood and also provide information on the recovery process for a brain injury such as the stroke.<span style="mso-spacerun: yes;"> </span>The way these researches went about their experiment involved four different procedures. Investigations were done for the biological and environmental factors, this was more of an observational study which involves a simple task such as book keeping, and keeping an open eye for changes that stood out. Some of these details that were compared to each other were for example the size of the specific injury, the location, the rate this injury occurred at and how fast these injuries took to recover. For the responses to injury section and the adaptive responses experiments were done on animals to compare the variety of responses the brain had to different scenarios. Cerebral edema is an example of the most common response, which is the excess accumulation of water in the intra/extra spaces in the brain. These responses were also compared in different areas, how fast they took to respond, how beneficial these responses were, and how they react to a variety of drugs. Taking to note that out of all the drugs that were used the two that are normally prescribed now on humans, actually were the ones that slowed the recovery process even more, these two were phenytoin and benzodiazepines. The third section also involved rats mostly adult rats. The neuronal rearrangements dealt with the new connections our neurons and axons make after an injury occurs and the connections that are lost. Some of these connections appear to happen 8 hours after an injury which would be beneficial if these connections would help, but they seem to impede the recovery process. In the other hand the connections that would be beneficial are of course the ones that take months to happen.<br />
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<br />
There are many drugs out there at this very moment that seem to aid the injury, however none stand out from the rest. As a person interest in neurology and someone who had her grandpa suffer such an injury I actually found this article very interesting, and even if you don't think this would interest you try and learn a little about it. I believe we all can agree the brain is the reason we function making it very important, but yet we still do not understand how it truly works.<br />
<br />
Here is the link if you are interested in reading more.<br />
<a href="http://stroke.ahajournals.org/content/21/11/1636.short">http://stroke.ahajournals.org/content/21/11/1636.short</a><br />
<br />
<strong>Citation</strong><br />
Goldstein, L. B., & Davis, J. N. (1990). Current Concepts of Cerebrovascular <br />
Disease and Stroke. <i>Restoratiave Neurology: Drugs and Recovery Following </i><br />
<i>Stroke</i>, <i>21</i>, 1636-1640. doi:10.1161/01.STR.21.11.1636 </div>
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</span>cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-17685260509692886672012-11-30T14:39:00.000-08:002012-11-30T14:41:26.905-08:00Decisions, Decisions <!--[if gte mso 9]><xml>
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My meeting with Amanda was great! I found out she was the
reason I enrolled in physics back in high school due to the great comments
other students told me about<span style="mso-spacerun: yes;"> </span>her, but
was surprised when I found out she had a new job and wouldn’t be teaching at
Maryvale High School anymore. It’s funny the reason I finally meet her is
because of this scholarship. Well our small chat was very informative and
helpful for me, she assured me that I am on top of my classes and my career
plan is right so far. Hearing this sure made my day, and made me feel like I do
not have to worry about questioning my advisor anymore.</div>
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The next day I was able to meet with Matt regarding my
internship hours. We talked about the hours I would be available to start my
internship and concluded how we might need to check our schedule every day to
make sure we can both work with my unstable schedule. <span style="mso-spacerun: yes;"> </span>I then I started to realize that the reason
why my schedule varies so much throughout the internship is because of my poor time
management. I am aware that there are small seminars, and public speakers that
take place here at Phoenix College and focus on this topic specifically. I
always thought to myself that it is not meant for me, I have always felt comfortable
making my own schedule and independently coming up with the best way to arrange
my responsibilities. However after that day, I feel like the only way to
becoming a successful S-STEM Scholar and at the same time enjoy my time as an
intern is to give up something I simply cannot do anymore. This week I talked
to Amanda and we came to a conclusion; I am now denying my federal work-study
award to fully receive my scholarship and to spend my time focusing on school,
and my internship.<br />
<br />
<br />
This picture is funny because for every minute there is something to do. Reminded me of my schedule. <br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg2xIvFRLS8asCYdMT1onRSBFwWIphQYM0usGL6HN9pl-Lz7bth67xq2shkbHXCa230UjX1UvxD0-Cdgxs_LeXLV1rE0JQK2TGOGOeB-dO85keIGygxbGxk_6Wshp2sNydNe8C4x-6gianR/s1600/Timemanagement.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg2xIvFRLS8asCYdMT1onRSBFwWIphQYM0usGL6HN9pl-Lz7bth67xq2shkbHXCa230UjX1UvxD0-Cdgxs_LeXLV1rE0JQK2TGOGOeB-dO85keIGygxbGxk_6Wshp2sNydNe8C4x-6gianR/s1600/Timemanagement.jpg" height="198" width="200" /></a></div>
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cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com0tag:blogger.com,1999:blog-3762715904401374837.post-56629508256655714382012-11-09T14:12:00.001-08:002012-11-09T17:29:10.218-08:00Hello Everyone !<br />
<br />
My name is Melina Calles, I am working on my Associates in Science here at Phoenix College, and plan to transfer to ASU to get a bachelors in Biological Sciences. I do not know what to expect from this opportunity but hopefully some great experiences ! <br />
<br />
Excited to see how this works ! <br />
<br />
Melina cmelinahttp://www.blogger.com/profile/11747916336941937073noreply@blogger.com1