GBS MasterMix Prep and QC protocol
Work in progress. This probably needs 2 for the 1st and 2nd PCRs.
MasterMix (make for 40 plates in 50mL conical tube)
Reagent | ul/rxn | rxns | ul needed (x1.4) |
|---|---|---|---|
H2O | 9.52 | 770 | 10,272 |
5x iProof buffer | 4 | 770 | 4316 |
10 mM dNTPs | 0.4 | 770 | 432 |
50 mM MgCl2 | 0.4 | 770 | 432 |
5 uM Illumina Primers | 1.33 | 770 | 1435 |
iProof TAQ | 0.2 | 770 | 216 |
DMSO | 0.15 | 770 | 162 |
total | 16 | 770 | 16,833 |
Aliquot 4,400 ul into 10 5ml tubes and label each #AmpMM_mm-dd-yy_#
Aliquot 16 ul into each well of an 8 pcr-tube strip. Add 4 ul TE to each well.
Cap, Vortex, and spin down. Run on thermocycler program GBS1:
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 30X | 0:30 |
60 | 30X | 0:30 |
72 | 30X | 0:40 |
72 | 1X | 10:00 |
4* | 1X* | 0:00* |
No cleanup needed. Run resultant rxns on a D1000 High Sensitivity Tape.