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.