Today we performed PCR using ISSR primers on previously extracted Lupinus arboreus samples. ISSR stands for interspersed simple sequence repeats, which are microsatellites. Instead of using the microsatellites as the markers, we use them as primers to generate fragments of DNA through PCR. We will then compare the length of these fragments to better understand population genetics of Lupinus arboreus.
First we each received 5 samples. Then, for each sample, we made three 1:10 dilutions so that each table could work with each sample. Additionally, PCR for these ISSR primers works better with diluted gDNA.
My samples and ISSR primer
Every table worked with the same 60 samples, but each table used a different ISSR primer. My table worked with the primer HB10. I also included a negative control. Below is table showing my samples and their corresponding label on their PCR tube. I also labeled each strip with my intials, AJC, and HB10.
Vanessa prepped the master mix as shown below, working on ice. Except Prof Paul added the taq at the end just before we add the master mix to our PCR tubes and put them in the thermocycler. Vanessa made enough Master Mix for 80 samples (our table had 64 total)
|Material||per rxn (µL)||per 80 rxns (µL)|
|10x buffer + Mg||3.00||240|
We first prepped our PCR tubes by adding 1 µL gDNA to each appropriately labeled tube. Then we added 19 µL of master mix to each tube. I pipetted up and down to mix each tube. Samples were kept on ice until they were placed in the PCR machine and ran.