Extractions !

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.

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.

On Tuesday I extracted DNA from the seven cultures I made last week. I noticed that the S. epidermidis 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.

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.

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.
   

Figure 1.
.5% Agarose Gel
Ruler
E. coli
S. aureus
B. subtilis
S. enterica
P. stuartii
S. marcescens
 

Figure 2
1% Agarose Gel
Ruler
E. coli
S. aureus
B. subtilis
S. enterica
P. stuartii
S. marcescens






Figure 3
1% Agarose Gel
Ruler
E. coli
S. aureus
B. subtilis
S. enterica
S. marcescens
P. stuartii