Lab 12 Jason Krastins

On November 19th, the Pyrimidudes started our lab session. The first thing we did was a PCR to test for successful library construction our Mimulus guttatus samples. To begin, we labeled 8 PCR tubes from 9 to 16. After, we prepped a master mix for RADseq with 11 reactions using 88uL of NEB One-Taq 2x Master Mix, 4.4uL of forward primer, 4.4 uL of reverse primer and 68.2uL of DNA-free water. After that, we each together added 1uL of each library DNA template to their corresponding tubes along with 15uL of master mix and ran PCR using the “PCR1” on te BIORAD #1/2. Next, we each loaded 2 of the products per person in the group ran the products of PCR1 for each sample on a 1.5% agarose gel with a 100bp ladder at 130V for 40 minutes. Then, we collected the gel. For the next PCR run of the day, we added the second barcode sequence along with the Illumina primers to our libraries of Mimulus guttatus, allowing us to identify which individuals. We yet again labeled 8 tubes from 9 to 16, then, we together prepared a master mix with 11 reactions using 3.40uL of Phusion DNA polymerase, 68.80uL of 5x Phusion HF buffer, 17.20 uL of 10uM PCR 2-5 forward primer, 17.20uL of 10uM PCR 2-5 reverse primer, 6.90 uL of 10mm dNTPs, 10.30 uL of DMSO and 118.30 uL of pure water. For the final step, we added 3uL of each template DNA to their corresponding tubes along with 22uL of master mix solution to each tube. We finally vortexed, then spun down the tubes in microcentrifuge and ran “PCR2” on BIORAD #2. To finish off the procedure, I ran 2uL of the products of PCR2 on a 1% agarose gel with a 100bp ladder, which was taken by Prof. Paul and stored.

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