A slight change in electrophoresis gel creation methodology yielded the clearest DNA banding I have yet received during this project. I have tested different gel recipes to-date, with varying results, and recently I began testing the optimal conditions for adding the DNA stain SybrGreen directly to my uncongealed gel solution before pouring and setting the gels. Previously, I simply added the DNA stain into each well as I added the DNA sample for testing; however, these gels showed limited or zero presence of DNA banding. To test the hypothesis that a direct addition would result in improved gel performance, I conducted a comparison experiment of the two gel types. I prepared four DNA samples using an identical protocol. I then created two gels from the same buffer/agarose base. I poured one gel, added DNA stain directly to the remaining mixture, then poured the second and allowed both to set. Finally, I loaded the samples. After thirty-five minutes running @200 volts, gel one showed zero DNA present, yet gel two showed clear banding. While this result was expected, it was exciting nonetheless.
Gel protocol:
The gels utilized for this test were created using a 0.9 g agarose/100 mL buffer mixture heated for 1:45 minutes on high in the microwave. Immediately after heating, gel #1 was poured. The remaining 75 mL mixture was allowed to cool to exactly 60°C, and then 3µl of 10,000x concentration SybrGreen was added and diffused into solution using a five-second swirling action. Gel #2 was poured, then both gels were allowed to set for 20 minutes in darkness (to prevent degradation of the SybrGreen due to UV light). The buffer mixture contained 980 mL/20mL de-ionized water/50X TAE buffer stock.
I was also able to expand the project to include additional bacterial species, which was another turn towards progress as my original hypothesis theorized the existence of universal primers that can be applied to multiple bacterial species. To-date, all experiments have utilized only E. coli. With this experiment, Pseudomonas aeruginosa, Proteus vulgaris, and Staphylococcus aureus were also subjected to extraction and gel testing. All four species yielded positive results after electrophoresis, with the gram-positive S. aureus providing the most limited results. These were the results I both projected would occur and hoped for. After some additional testing to replicate this week's results with the added bacterial species, this project should now be able to move forward to the next phase, which is intended to focus on primer design.
Altogether, it was a successful week. Please enjoy this photo of my gel results until the next blog. Cheers!
Left to right: E. coli, P. aeruginosa, P. vulgaris, S. aureus
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