Hi everyone! So unfortunately, our third attempt at PCR didn’t work… and neither did our fourth attempt. We started our fifth attempt this morning (and we’re going to end up running over 90 tubes this attempt!) so let’s hope we get something this time. After our third round of PCR didn’t work, we hypothesized that we might not have enough genomic Anopheles DNA, so we decided to use another group’s DNA that we knew worked. At the same time, we decided to raise the extension times and play with the annealing temperatures to try to get a band on our gels. To be able to move on to the next step of this process, ligation, we would have needed to see bands by last Monday. Since we didn’t see anything, we knew we wouldn’t be able to continue on and finish everything by the end of the quarter, so we decided to just focus on troubleshooting our PCR. We decided that since we’re just going to focus on PCR we would also troubleshoot Canoe, in an effort to get SOMETHING. Unfortunately, at this point we still haven’t gotten any bands on our gels, which makes us think there may be something wrong with the primers that we’re using to extract the genes. This weekend we’re going to try running a variety of semi-nested PCR reactions to test to see if any of the primers are defective. Had our PCR worked, as most groups’ did, we would have ligated our PCR product into plasmid vectors, and then transformed these vectors into competent E.Coli bacteria, similar to what we did in Superlab last quarter with our bacterial DNA. After transformation, we would have prepared mini-preps of the DNA from bacterial cultures, sent the samples away to be sequenced, and then analyzed the sequences to determine if they were what we were expecting to find. If our PCR this weekend works, we may still be able to ligate and transform, but unfortunately we’re so close to the end of the quarter we won’t have time to send it away for sequencing and get it back before the end of next week, which is disappointing, but that’s science! This coming Friday, we’re going to give in-class presentations about what we did this quarter and our results, so we anticipate our presentation having a LOT of PCR!
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