November 19, 2019
Test PCR 1
To begin our lab, we tested for successful library construction of our Mimulus guttatus samples (restriction digest and ligation of barcodes) using a test PCR. We first labeled 8 PCR tubes with the sample ID# 17-24. I then prepared a master mix for RADseq which contained; 88μL of NEB one-taq 2x Master Mix, 4.4μL ofForward Primer (PCR1 X), 4.4μL of Reverse Primer (PCR2 6), 68.2μL of pure H2O, and 1μL of the library DNA template. I added the 1μL of each library DNA template to their corresponding labeled tubes and added the 15μL of Master mix as well. The PCR was run using the “PCR1” on BIORAD #1/2. Next, the products of PCR 1 were ran for each sample on a 1.5% agarose gel with a 100 bp ladder at 130V for 40 minutes.
Test PCR 2
In the second part of our lab, we completed a second PCR run. First, we had to ass the special second barcode sequences and the illumina primers to our Mimulus guttatus libraries. This allowed us to identify which specific individuals a given sequence comes from. I started off by labelling 8 tubes with the sample ID# 17-24. We then moved on to prepare the master mix which contained; 3.4μL of Phusion DNA polymerase, 68.8μL of %x Phusion HF buffer, 17.2μL of 10μM PCR 2-5 forward primer, 17.2μL of 10μM PCR 2-5 reverse primer, 6.9μL of 10mm dNTPs, 10.3μL of DMSO and, 118.30μL pure H2O. After the master mix was made, I added 3μL of each template DNA to its corresponding labeled tubes. In addition, I added 22μL of the master mix. The tubes were capped, vortexed, and microcentrifuged. The PCR was run using the “PCR2” on BIORAD #2. Lastly, 2μL of the products from PCR2 were ran on a 1% agarose gel with a 100bp ladder.