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.
On 11/5/19, the Pyrimidudes 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. After this, I added 6uL of each of my samples’ DNA to each tube. Next, we worked together to prepare 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. Each of us then added 3uL of master mix to each of the 2 samples we were responsible for and sealed, vortexed, and centrifuged. After that, Prof Paul put the tubes in the incubator at 37 degrees celsius for 8 hours.
The next lab period on 11/12/19, we started our second task, which was doing adapter ligation for RADseq. I added 1uL of the working stock EcoRI adapters , EcoRI_8 and EcoRI_9, to my samples with sample IDs 13 and 14. The Pyrimidudes 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, we added 3uL of master mix to the digested DNA and sealed, vortexed, and centrifuged the tubes. Prof Paul then incubated the tubes at 16 degrees celsius for 6 hours.
Last Tuesday, my group, the Pyrimidudes, started our lab session which involved doing PCR reactions to amplify the DNA we gathered by running gel electrophoresis on our M. guttatus tissue samples gathered the Tuesday prior. 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; D1, D2 and D3, and added 19 ul of the above master mix along with 1uL template from JK CATB 01, and ALC TROJ02, and ALC SHOR 003 to each tube respectively. Finally, I vortexed the tubes and stored them in ice until Dr. Paul could put them in the PCR machine to amplify the DNA for processing next lab period.
Last Tuesday, the Pyrimidudes ran a gel electrophoresis of our Mimulus guttatus DNA samples that were amplified last week. First, I dotted out 13 loading dye dots on a sheet of parafilm. Then, each member of our group I pipetted 3 uL of each of our individual 3 samples of our PCR products into its own dot, pipettied up and down to mix, and then loaded all dots into the gel. After all members had added their DNA plus the ladder, Dr. Paul ran the gel at 130 volts for 30 minutes, then stored the products in the freezer for future use.