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.

On Tuesday, 11/12/19, we started our lab session. Our first task was to conduct double-digest restriction associated DNA sequencing. I labeled 2 tubes as P5 and P6, for my samples CHIM 002 and PRBM 005. Then, I added 6uL of each of my samples’ DNA in the corresponding tube. We prepared a master mix with 11 reactions using 9.9uL of CutSmart buffer 10X, 3.1 uL of EcoRI-HF enzyme, 1.3 uL of MSPI enzyme and 18.7 uL of pure water and we placed the master mix on ice. I added 3uL of master mix to each sample and sealed, vortexed, centrifuged and incubated the tubes at 37 degrees celsius for 8 hours. After that, we started our second task, adapter ligation for RADseq. I added 1uL of the working stock EcoRI adapters , EcoRI_8 and EcoRI_9, to my samples with sample IDs 15 and 16. We prepared an adapter ligation master mix with 11 reactions using 4.4 uL CutSmart buffer 10X, 14.3uL ATP(10mM), 2.2 uL T4 Ligase, 1.1 uL pure water. Then, I added 3uL of master mix to the digested DNA and sealed, vortexed, centrifuged and incubated the tubes at 16 degrees celsius for 6 hours.

On Tuesday 11/05/19 at 2:30 PM, we started our lab session which involved PCR reactions. We prepared a master mix using 214uL of ddH2O, 32uL of 10x buffer, 32uL of MgCl2, 16uL of BSA, 3.2uL of dNTPs, 3.2uL of F-primer, 3.2uL of R-primer and 1uL of Taq. Then, I labeled 4 tubes with the following tags; D4, D5 and D6 and added 19 ul of master mix along with 1uL template from EO cATB and JP MW 03 and SHOR 004 to each tube respectively. Finally, I vortexed the tubes and stored them in ice.

On Tuesday, 10/29/19 at 02:20 pm, we started to undergo gel electrophoresis of our mimulus DNA samples from last week. First, I dotted out 16 loading dye dots on a sheet of parafilm. Then, I pipetted 3 uL of each of my PCR products into its own dot and loaded all dots into the gel. Finally, I ran the gel at 130 volts for 30 minutes.