November 12, 2019
Double Digest
In our lab, we set out to digest our Mimulus guttatus samples by beginning our double-digest restriction associated DNA sequencing. I labeled 8 PCR tubes (1-8) that contained 6μL of sample each and stored the tubes on ice. We then prepared a master mix that contained; 9.90μL of CutSmart buffer 10X, 3.1μL of EcoRI-HF enzyme, 1.3μL of MSPI enzyme and 18.7μL of pure H2O for a total of roughly 33.0μL. The master mix was capped in the tube, mixed well, centrifuged, and stored on ice. I then added 3μL of master mix to all 8 PCR tubes with the DNA samples. The tubes were sealed, vortexed, placed in the centrifuge and incubated at 37° C for 8 hours on a thermocycler with a heated lid set to 50°C.
Ligation
Next, we were given the task of completing an adapter ligation for RADseq. Before making the Master Mix, I added 1μL of the working stock EcoRI adapter, EcoRI 8 and EcoRI 9, directly to my digested DNA samples which were sample ID# 23 and 24. I was then able to create the adapter ligation master mix for RADseq. The master mix contained; 4.4μL of CutSmart buffer 10X, 14.3μL of ATP, 2.2μL of T4 Ligase, 1.1μL of pure H2O and 11.0μL of Universal P2 MSPI adapter for a total volume of 33.0μL. After it was complete, I added 3μL of the master mix to the digested DNA. The tubes were sealed, vortexed, placed in the centrifuge, and incubated at 16°C for 6 hours on a thermocycler with a heated lid set to 50°C.
