...
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 | 2681800 | 276117136 |
5x iProof buffer | 4 | 2681800 | 11607200 |
10 mM dNTPs | 0.4 | 2681800 | 116720 |
50 mM MgCl2 | 0.4 | 2681800 | 116720 |
5 uM Illumina Primers | 1.33 | 2681800 | 3852394 |
iProof TAQ | 0.2 | 2681800 | 58360 |
DMSO | 0.15 | 2681800 | 43.5270 |
total | 16 | 2681800 | 464028800 |
Add 16 ul of PCR1 MM to each well of four 2+12(for 4Trout) new hard shell PCR plates with 8 channelAssistPlus program 16ulMMDisp on 125ul ViaFlo
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:
Temp C | Cycles | Time |
|---|
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* |
...
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 | 2721800 | 136765 |
10 mM dNTPs | 0.4 | 2721800 | 128720 |
Primers | 1.33 | 2721800 | 4262394 |
Total | 2.155 | 2721800 | 6903879 |
Then continue with the last four unlisted steps for GBS1 from above:
Temp C | Cycles | Time |
|---|---|---|
98 | 1X | 3:00 |
60 | 1X | 2:00 |
72 | 1X | 10:00 |
4 | 1X | 0:00 |
Template | EcoR1 MID plate | Pool Plate | Library | Norm to | Sequencer |
|---|---|---|---|---|---|
AR1 | 5 | 1+2 | 1 | 10 | Nova |
AR2 | 6 | 1+2 | 1 | 10 | Nova |
AR3 | 7 | 3+4 | 1 | 10 | Nova |
AR4 | 8 | 3+4 | 1 | 10 | Nova |
Size Select at 275-375 bp on Pippin Prep 2% no dye gel