...
Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate. Leave out at room temperature for ~2 hours.
PCR1:
Reagent | ul/rxn | rxns | ul needed (x1.4) |
|---|---|---|---|
H2O | 9.52 | 1800 | 17136 |
5x iProof buffer | 4 | 1800 | 7200 |
10 mM dNTPs | 0.4 | 1800 | 720 |
50 mM MgCl2 | 0.4 | 1800 | 720 |
5 uM Illumina Primers | 1.33 | 1800 | 2394 |
iProof TAQ | 0.2 | 1800 | 360 |
DMSO | 0.15 | 1800 | 270 |
total | 16 | 1800 | 28800 |
...
Add 4uL of template from pooled plates with Benchsmart or Assist Plus PoolWINPlatePCR on 12.5 ul ViaFlo (This pools 4 samples together with each sample being pooled with 2 separate sets of samples)
Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:
...
Add 2.155 ul of the MM directly to the previous PCR as soon after sample reaches 4C as possible
Reagent | ul/rxn | rxns | ul needed (x 1.6) |
5x Iproof buffer | 0.425 | 1800 | 765 |
10 mM dNTPs | 0.4 | 1800 | 720 |
Primers | 1.33 | 1800 | 2394 |
Total | 2.155 | 1800 | 3879 |
...
Size Select at 275-375 bp on Pippin Prep 2% no dye gel