On Wednesday Chris Smith kindly showed us how to use fluorescence to determine how much mitochondrial DNA we amplified last week. This is done on a Quantitative PCR (qPCR) machine using picogreen.
When we ran our reaction, both long products and unintended smaller pieces amplified. We needed to separate the unwanted smaller products out from the longer ones. To run all nine samples using four primer pairs, we had to run two gels. Gels use charge to push DNA through the gel. DNA is negatively charged, so applying a negative charge forces the DNA to move through the gel. Bigger pieces of DNA will get stopped in the gel earlier and smaller pieces will travel further, so when looking at the gel after it is finished running, we can determine which bands are bigger pieces of DNA.
I enjoy making and running gels because we can finally see what we are dealing with. We mixed 1 g of Agarose in TAE, and then heated the mixture up in a microwave. Apparently Agarose is quite viscous and will burn you and refuse to come off if it touches your skin, so Heather kindly didn’t let us handle it while it’s heating. After the Agarose dissolved we poured the gels and learned the hard way to not assume that they’re set before trying to yank them up.
Unlike yesterday, we ran the gels at the recommended 60 mV so that our bigger chunks of DNA wouldn’t be pushed through the gel too quickly or get heated up and damaged. After an hour we read the gels under UV light and they both showed DNA, which was a good sign.
However, we still needed to get the DNA out of the gels. The Gel-Doc has a UV light underneath the tray that the gel sits on, so the DNA bands are illuminated. We cut the brightest bands (the DNA) out with a razor and put them in a tube. This was dicey because we had to wear sunglasses and a scratched or damaged UV shield so that the UV light didn’t burn us up. This made spatial reasoning more difficult. The bands were completely surrounded by the gel, so we had to cut the gel away from all six sides of the band while moving quickly enough so that the DNA wouldn’t be damaged by the UV light. While one person cut out a band, another would tare a labeled tube and then hold it out for the cutting person to put the band in, then weigh and record the tube, and finally return for another band and repeat the process. We had an exchange of tare puns with Peter’s group. There were some terrible ones and we finally had to tear ourselves away.
After we got the bands in the tubes with nobody burned from the UV light, we added a Guanidium buffer (more Guanidium! Yay!) to each tube and centrifuged the tubes. We ended the day by eluting the DNA cut from the gels and loading the qPCR machine. Unfortunately, we failed to retrieve the data but the next morning Chris saved the day and exported our data. Luckily the data was there; we had just messed up getting it off the computer. Unfortunately, the data wasn’t great: some of the samples that should have had similar values were drastically different and our standard curve was not a nice line. We figured that our pipettes were off and got permission to use Chris’s pipettes for the next time. We re-ran the picogreen assay and got improved results: our standard curve had an r^2 of 0.89 and the values were more similar across primers. Primers A-D had reasonable amounts of DNA but Primers E-H had extremely low amounts.