Troubleshooting
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!