Today’s experiment: start the realtime experiments with my cell samples
Well, I’m glad I ended up starting my cell samples a little early.
These last few days I’ve been learning how to use a new machine, which I’ll try to post an image onto this blog for Day 19. But to get data for Day 18, I decided to run an experiment on my cell samples, which I wasn’t planning on doing for a little while.
Except it turns out that my primer stocks got contaminated.
Realtime PCR (sometimes referred to as “qPCR” for “quantitative PCR”) takes a small amount of DNA, uses short DNA sequences called primers to attach to that DNA, and the PCR reaction amplifies a section of DNA that you want to amplify. Primers include forward and reverse primers, meaning that during the reaction the only region of DNA that will be amplified is between these two primers. In the plate I ran yesterday, I used primers specific for 2 genes that I wanted to amplify and measure.
As part of the qPCR controls, I had a no template control (NTC) well for each of the genes, which should get no amplification (except for maybe primer dimers, which happen when the forward and reverse primers anneal to each other, but often this is a small chance and doesn’t affect the result). But this gel shows me that I have amplification in both of my NTC wells (the last 2 lanes), which means that some DNA somewhere got into my primers stocks. So I’m ordering more, they should arrive in the next week or so.
So that experiment’s on hold til the primers arrive. I’m going to go mess with this new machine now 😀