The next day after finding out that the PCR did not work, it was time to test a different protocol. This protocol is called "Isolation and Purification of Total Genomic DNA from E Coli", an experiment that is longer compared to my first experiment.
The procedures for this experiment began just like the first experiment. I had to grow bacteria over night for 24 hours in a TSB tube, but since I had my bacterias already growing from the first experiment I went ahead and used the same ones (E. Coli, S. Aureus, S. Epidermidis, and Salmonella). My S. Epidermidis tube was spilled when transferring so I used a different TSB tube that was grown a week earlier. I had to do some centrifuging at high speeds for intervals of 2-3 minutes. As well as some incubating at 37-80 degrees Celcius for 30 minutes and 5 minutes. Lysis solution was added to disrupt membranes and denature proteins. RNase solution was added to degrade RNA into small fragments or ribonucleotides. Protein Precipitation Solution to add insoluble material for the centrifuging step. Isopropanol was added to the solution, also 70% ethanol, and lastly the DNA Rehydration Solution (TRIS EDTA Buffer). In the end of all these procedures the extractions made were ready to run in either techniques. The first one I did was the gel extraction, the gel was made out of 100 milliliters of TAE and 1 gram of molecular Biology Agarose. When analyzing the gel under uv light, I came to the conclusion that this extraction only worked for the E. Coli and not for the rest. Once again a PCR was not done because I had no DNA from the extraction to run. Here are some pictures enjoy !
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| Closest to 80 degree Celsius the incubator reached. |
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| Gel Extraction Apparatus |
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| TAE Solution |
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