DD-RADseq (Test PCR+Final PCR)

On Tuesday, 11/19/19, we started our lab session. Our first task was to conduct a test PCR to test for successful library construction our Mimulus guttatus samples. We first labeled 8 PCR tubes from 9 to 16. Then, we prepared 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 pure water. After that, I added 1uL of each library DNA template to their corresponding tubes along with 15uL of master mix and ran PCR using “PCR1” on BIORAD #1/2. Next, I ran the products of PCR1 for each sample on a 1.5% agarose gel with a 100bp ladder at 130V for 40 minutes. We moved to the second phase of the lab as we completed the test PCR. For the next PCR run, we had to add the special second barcode sequences and the illumina primers to our libraries of Mimulus guttatus, allowing us to identify which specific individuals a given sequence comes from. First, I labeled 8 tubes from 9 to 16, then, I 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. After that, I added 3uL of each template DNA to their corresponding tubes along with 22uL of master mix. I vortexed and spinned down the tubes in microcentrifuge and ran “PCR2” on BIORAD #2. Finally, I ran 2uL of the products of PCR2 on a 1% agarose gel with a 100bp ladder.

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