Lab 11

For today’s lab we did DD-RadSeq of our samples of M. Gattatus plus some samples taken by Dr. Paul’s research Lab.

For Dr. Paul’s samples:

1. Adapters were thawed out on ice.
2. Add 1µL of working stock of EcoRI adapter straight into the digested DNA
3. Create a master mix at 130% needed volume with the following:
   1. 0.4µL CutSmart Buffer 10x
   2. 1.3µL ATP 10mM
   3. 0.2µL T4 Ligase Enzyme
   4. 0.1µL Pure H20
   5. 1.0µL Working stock of universal P2 MspI adapter
4. Add 3µL of the master mix to digested DNA
5. Centrifuge and incubate at 16˚C for 6 hours.

For our samples:

1. Place 6µL of each sample’s DNA in a PCR tube and store on ice.
2. Prepare a master mix containing the following per each reaction in a total volume of 3µL:
   1. 0.9µL CutSmart 10X Buffer
   2. 0.28µL EcoRI-HF enzyme
   3. 0.12µL MPSI enzyme
   4. 1.7µL pure H20
3. Because there are very small volumes needed for the master mix, a total volume of 130% was made to compensate.
4. 3µL of master mix was added to each labeled PCR tube.
5. Samples were then centrifuged  and incubated at 37˚C for 8 hours on thermocycler.