This week, I decided that in order to really move forward with my project, I had to be certain that the techniques I have been using are viable. Of course, one of the best ways to verify results is to simply repeat them using different variables. So I decided to test my extraction and electrophoresis gel techniques by applying them to additional gram-negative bacterial species. You may recall that to-date I have been working solely with E. coli; this week, I expanded my research to include Salmonella enterica, Shigella sonnei, Proteus vulgaris, Proteus mirabilis, Klebsiella oxytoca, Providencia stuartii, and Serratia marcescens.
I conducted extractions on all eight species, including E. coli, using the isopropanol method I have posted on here previously. I then ran two electrophoresis gels on the eight extraction samples, using the gel production method that fellow scholar Matt Hill and I developed last semester. Both techniques had demonstrated positive results in previous experiments. The results this time were gratifying; all eight samples showed positive results for DNA banding. (picture below)
This was extremely encouraging news, so I decided to push my luck and also test my polymerase-chain reaction (PCR) protocol and the primers from the first phase of my study. Despite my best efforts, I had been unable in phase 1 to achieve DNA amplification during a PCR.
The result? Good news! Three of my eight species were positive for banding post-PCR. This is a desired result, because it proved that my basic techniques were viable through that point. And because the results were not positive for all tested species, they also indicated that the primers selected were not universal. In other words, I can move forward with the next phase of my research: identifying a sequence of genes, in each bacterial species, that is similar; and developing a set of primers that will successfully target that gene sequence.
As we say around the lab, "There are no wrong answers in science, only new directions." Now, with the results of this experiment, I know what direction I need to go in next.
Until next week, enjoy!
Gel, pre-PCR, eight species sample. Source: Paul C.
Excellent work. Have you considered extracting the DNA from the strain of Ecoli used in the transformation lab and comparing it to see if there is a difference in the PCR products of the transformed and non-transformed Ecoli? If there is, and you can produce results the support that, it may be a useful tool that could potentially be used as a follow-up student lab activity. It does not seem as though testing that particular research question should take more than a week or so to complete. Just a thought...
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ReplyDeletegiven our conversation earlier this week, you should also click on the following link... Enigma
ReplyDeletecould you also post the ACTT # for the bacteria listed in your blog? Have you considered submitting your research to a journal for publication
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