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.