Thursday, April 25, 2013

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


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