This lab, we performed a DDRadseq of Mimulus guttatus samples. DNA samples collected from various locations (see Table 1 for codes) were chosen and 6 uL of each sample was placed in a PCR tube and stored on ice. A master mix was created with 9.9 uL of CutSmart 10x bugger, 3.1 uL ECORI-HF enzyme, 1.32 uL of MPSI enzyme, and 18.7 uL of pure H2O. 3 uL was added into each sample’s DNA. Samples were sealed, vortexed, and then placed in an incubator at 37 degrees Celsius for 8 hours on a thermocycler with a heated lid set to 50 degrees Celsius.
Our previous samples, prepared in the same way above, were then used to perform the second step of DDRADseq. Samples were placed on ice and 1 uL of EcoRI’s were added (see Table 2) to each sample. A master mix was created with 4.4 uL of CutSmart buffer 10x, 14.3 uL of ATP 10 mM, 2.2 uL of T4 igase enzyme, 1.1 uL pure H2O, and 11 uL of working stock universal P2 MspI adapter. 3 uL of this master mix was added to the digested DNA. Samples were sealed, centrifuged, and incubated at 16 degrees Celsius for 6 hours on a thermocycler with a heated lid set to 50 degrees Celsius.