Today, I worked with Sam to do a trial qPCR assay using some old juvenile oyster samples from a graduate student’s experiment. We used 8 cDNA samples (4 control, 4 CO2 exposed) and looked at 3 different genes: elongation factor, HSP90, and CYP1A. Sam showed me the calculations for creating a 15 microliter master mix and I created master mixes for the 3 gene targets. For each master mix we added: 5′ primer, 3′ primer, qPCR master mix, and water. I’ve attached a picture of our calculations below:
We decided to multiply each volume by 8.8 (10% greater than the actual needed amount) in order to ensure that we had enough master mix. I then transferred 15 microliter of each master mix to the well plate along with 5 microliter of cDNA sample. 3 negative control samples were also kept on the plate in which I put 5 microliter water instead of cDNA. Note: we initially forgot to account for the negative control samples in our master mix calculations, so we had to go back and add additional master mix volume to the original solution (which is what the red numbers on the right indicate).
We then took the well plates to the qPCR machine where Sam walked me through the protocol for the amplification phase as well as the theory behind it. We left the samples running (the machine stated that it would take approximately 1hr 30 minutes to complete) and will check the curves when I come in to the lab next.
Samples were kept on top of the box labelled 1 in the 2nd freezer: