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Tuesday, July 13, 2010

PCR Troubles

Troubles with PCR is nothing new to the interns working in the DNA Disco center. Team Venom was notorious for having contamination. Gel after gel would reveal that their PCRs were contaminated with some unknown source. A possible reason for why their contamination was so frequent was that their venom gland tissue samples were somewhat old and came pre-contaminated or that their primers were not the good enough quality for their annealing temperature in the PCR process. To try to prevent more contamination they bleached their bench space, changed to filtered tips, sterilized their pipettes, and switched their reagents. Since it is extremely difficult to pinpoint what caused the contamination, some untrustworthy feelings arose amongst the venom team. As a result of their numerous precautions, the problem of contamination has simmered down. However, for our team some problems were just coming to the surface.

Today we were ran gels of DNA from two separate genes and the gels revealed a conundrum. No bands on the gel image showed up. The bands represent the presence of DNA in the PCR samples and when no bands are visible, that means that there was human error during the process of PCR.

What a normal gel image should look like:
All bands are bright and visible, ladders (far left) showed up clearly, blank (circled in black) is empty, meaning no contamination.

What our faulty gel images look like:
Only ladders appear, no DNA bands visible.

The most plausible explanation for this absence of bands is that we forgot a reagent in the master mix of the PCR. We found this puzzling as we had not one but two different PCR's with this problem. Tomorrow we will be redoing these faulty PCR's and get feedback from our advisor, Kevin, who has been away at a conference for several days.

Hopefully we can resolve the issue and determine the exact source of our error.

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