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!
Archive for December, 2012
Happy December! The past couple weeks have been really stressful, what with Thanksgiving break and midterms for almost all of my classes, but I have some time to post again, now that the majority of my work is done (for now). A lot has been going on in Superlab lately. The day before Thanksgiving break, we set up our second round of PCR, which was nested PCR in an effort to extract only our gene. This past Wednesday, we ran all of our samples from both the first and second rounds of PCR on a gel (12 samples total) and imaged it to see if we got bands in the correct places. Unfortunately, our gel looked pretty strange. Heartless amplified a ton of DNA, so much that the band pattern looked like a DNA ladder (what’s used to determine the length of amplified DNA fragments), which looked nice but really wasn’t what we wanted to see. On the other hand, Canoe didn’t amplify anything, so we ended up striking out twice. Luckily, this isn’t unusual. Only one group in our lab section actually got a band at the correct size, so our situation isn’t too rare. We decided that for time’s sake we’re going to focus on Heartless, rather than trying to clone two different genes, since we were starting to go in so many different directions. We hypothesized that the strange amplification pattern observed was due to incorrect primer dilutions, so we re-made our stock primer solutions and re-ran the first and second rounds of PCR, this time using different temperatures and semi-nested PCR, which is similar to nested PCR but uses a different combination of primers. We ran those samples on a gel this morning, and, drum roll please… got nothing. We think something may have gone wrong with the first round of PCR such that no DNA was amplified, which is disappointing, but gives us a starting point for troubleshooting. I’m going back into lab in a little while to re-make our first round PCR reactions, and hope that the third time’s the charm! We’ll be able to run the second round of PCR tomorrow morning, and run all the samples on gels tomorrow night, so that if we get any product (fingers crossed!!) we’ll be able to start the next step of the process—ligation—on Monday afternoon. If we don’t get bands a third time, we’ll be able to troubleshoot more, and hopefully get something by Wednesday. At this point in the quarter, with only two weeks left before finals, we’re really getting down to the wire, so we really have to hope we get some usable DNA this week!