Poster time !

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.  I am still waiting on the few results I hope to include in my poster that were done this week.

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.  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.

Hind III enzyme
 

Bacteria :

• E. coli
• P. stuartii
• B. subtilis
• S. enterica
• S. marcescens
• S. aureus
• 

  

  Ecor 1

  S. epidermidis

RFLP Protocol

1. 10 ul buffer
2. 86 ul DNA
3. 4 ul of Ecor1 enzyme or Hind III enzyme
4. Incubated at 37 c degrees for 1 hour

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.   

Bacteria:

• S. marcescens
• S. enterica
• B. subtilis
• S. epidermidis
• E. coli
• S. aureus
• P. stuartii

 Primers

• 2 ul primer 3 + 38 ul TE
• 2ul primer 4 + 38 ul TE
• 2 ul primer 6 + 38 ul TE

PCR Protocol                                                                                                      

1. 10 ul master mix
2. 5 ul DI H2O
3. 4 ul primer (3, 4 or 6)
4. 1 ul DNA
   
    

 

Slowly but surely

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,  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.

DNA

• E. coli
• B. subtilis
• S. aureus
• Salmonella
• S. marcescens
• P. stuartii  

PCR Protocol

1. 10 microliters of master mix
2. 5 microliters of DI H2O
3. 4 microliters of primer (3,4,5,6)
4. 1 microliter of DNA 

Primer 3

Primer 4

Primer 6

Primer 7

RFLP Results using DNA extractions from 4-11-13

       RFLP Protocol

      1. 10 microliters of 1 x buffer

      2. 88 microliters of DNA

      3. 2 microliters of Ecor 1 enzyme

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.

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. 

Another week down, about three to go!

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^nd.

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.

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.  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

• S. aureus from the extractions done on 3-28-13
• S. marcescens from the extraction done on 3-28-13
• P. stuartii from the extraction done on 4-2-13

Primers                                                                                                   PCR Protocol

2ul of primer 3 + 38ul of TE H2O= Primer 3                                         1.       10ul of master mix

2ul of primer 4 + 38ul of TE H2O= Primer 4                                         2.       5 ul of DI H2O

2ul of primer 6 + 38ul of TE H2O= Primer 6                                         3.    4ul of Primer (3,4,6,7)

2ul of primer 7 + 38ul of TE H2O= Primer 7                                         4.       1ul of DNA



PCR Results using Primer 6 & 7
Ruler
S. marcescens primer 6
S. aureus primer 6
P. stuartii primer 6
S. marcescens primer7
S. aurues primer 7
P. stuartii primer 7
 

PCR Results using primer 3 & 4
Ruler
S. marcescens primer 3
S. aureus primer 3
P. stuartii primer 3
S. marcescens primer 4
S. aureus primer 4
P. stuartii primer 4
 

 

 



I 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!



QuickExtract Kit Extractions
Ruler
E. Coli
B. subtilis
S. aureus
S. marcescens
P. stuartii
Salmonella
S. epidermidis

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.

 



 

 

      

Ok so about two weeks ago I think you can recall that I was missing my Extraction Kit so I began extracting DNA from E.coli 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 E.coli. 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 E. coli 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!


Figure 1
RFLP using E.coli
Ruler, A, B, C

Tube	10x buffer	DNA	H2O	Hind 111	Ecor 1
A	2ul	1ul	17ul	0ul	0ul
B	10ul	50ul	32ul	8ul	0ul
C	2ul	2ul	17ul	0ul	1ul

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

1. 10ul of 1x buffer
2. 88ul of DNA
3. 2ul of Ecor1 enzyme

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.



RFLP
Ruler
S. marcescens (3-38-13)
S. marcescens (3-26-13)
S. aureus (3-38-13)
P. stuartii (4-2-13)

 

1. 1ul sybr green
2. 2ul orange loading dye
3. 8ul of DNA

1. 9ul molecular weight ruler
2. 1ul sybr green

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.