We were able to digest out Mimulus guttatus samoles with selected restriction enzyme. We also utilized a digestion buffer appropriate for both enzymes. We placed 6 microliters of each sample’s DNA in the well of a PCR tube, and stored it on ice. We prepared a master mix with 9.9 microliters of CutSmart 10x buffer, 3.08 microliters of EcoRI-HF enzyme, 1.32 microliters of MPSI enzyme, and 18.7 microliters of pure water. To account for the multiple round of pipetting in the viscous liquid, we added 130% excess mastermix to each sample’s DNA. The total reaction volume amounted to 9 microliters and then the samples were sealed, vortexed, centrifuged and incubated at 37 degrees Celsius for 8 hours on a thermocycler with a heated lid set to 50 degrees Celsius.
In order to perform this double-digest restriction associated DNA sequencing, it it necessary to address adapter ligation. We first had to thaw the working stock EcoRI and MspI adapters that were made previously. We added 1 microliter of the working stock EcoRI adapter directly to each tube of digested DNA as follows: sample BT1 had Eco_2, sample BT2 had Eco_3, sample RM1 had Eco_4, sample RM2 had Eco_5, sample GS1 had Eco_6, sample GS2 had Eco_7, sample AH1 had Eco_8, sampled AH2 had Eco_9. A master mix was prepared with 4.8 microliters of CutSmart buffer 10x, 15.6 microliters of ATP, 2.4 microliters of T4 Ligase, 1.2 microliters of pure H2O, and 12 microliters of universal P2 MspI adapter (E). 130% excess of the mastermix was added to the tubes with the digested DNA and working stock to accommodate multiple round of pipetting with the viscous nature of the glycerol of the enzymes. We added 3 microliters of the mastermix to the digested DNA.
The total reaction volume of 13 microliters were sealed, vortexed, centrifuged, and incubuted at 16 degrees Celsius for 6 hours on a thermocycler with a heated lid set to 50 degrees Celsius. The samples were then stored frozen.