I start each day at the qPCR machine in CIEMAS, picking up the data from last night’s qPCR run. A quick scan of the amplification graphs can usually tell me if there were any problems with the qPCR, but I’ll plot the results later to make sure.
I head over to BioSci next. The first thing I do whenever I enter the lab is greet Peabody, the lab manager’s puppy. He’s adorable and he loves people. Sometimes he likes to climb onto my lap while I’m working. I bet he’s paying attention to what I’m doing in case he decides to become a scientist when he grows up.
When I work with qPCR data, I start by plotting the standard curves, which are two sets of serial dilutions of DNA. I know what these plots should look like, because I know how much DNA is going into each well, and because I prepare these columns in exactly the same way for each plate. Here’s an example:
The standard curves are my best indication as to how reliable the rest of the plate is, so if they don’t look like the plot above, then I discard the plate and redo all of the samples.
Another way that I check the reliability of my data is by running duplicates of each sample. If the difference between the values for a pair of duplicates is greater than 1, then I discard both data points and redo the sample on another day.
As long as the data looks good, I add it to my master spreadsheet, which I am slowly filling up with results from the 225 samples in the project. Then, I usually have a few hours to read papers for my next project, practice coding, or learn about the statistical tests that I’ll need to analyze my data.
In the afternoon, I prepare another qPCR plate. This involves several steps; I have to select 20 samples and dilute them to the same concentration, prepare the two columns of standard macaque DNA for the standard curves, make a qPCR master mix, and transfer the correct combination of DNA, primer, and master mix into each of the 96 wells on the plate. With so many details to keep track of, it’s easy to make a mistake somewhere. That’s why I include the standard curves on each plate and run duplicates of each sample.
At the end of the day, I head back to CIEMAS to start the qPCR plate running! I’ll pick it up tomorrow morning.
