Should we not prep this MM ahead of time?
PCR Amplification
Pool | Plate1 | Plate2 | Plate3 |
|---|---|---|---|
3A | Trout8 | Trout9 | Trout10 |
Pool | Plate1 | Plate2 | Plate3 |
|---|---|---|---|
3B | Trout8 | Trout9 (FLIPPED) | Trout10 |
PCR1:
Reagent | ul/rxn | rxns | ul needed (x1.4) |
|---|---|---|---|
H2O | 9.52 | 192 | 2570 |
5x iProof buffer | 4 | 192 | 1080 |
10 mM dNTPs | 0.4 | 192 | 108 |
50 mM MgCl2 | 0.4 | 192 | 108 |
5 uM Illumina Primers | 1.33 | 192 | 359 |
iProof TAQ | 0.2 | 192 | 54 |
DMSO | 0.15 | 192 | 41 |
total | 16 | 192 | 4320 |
Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel
Add 4uL of template from pooled plates with Benchsmart
Seal, Vortex, Spin down, and then 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* |
*pause step
Extra PCR
Add 2.125 ul of the MM directly to the previous PCR
Reagent | ul/rxn | rxns | ul needed (x 1.6) |
5x Iproof buffer | 0.425 | 192 | 131 |
10 mM dNTPs | 0.4 | 192 | 123 |
Primers | 1.33 | 192 | 410 |
Total | 2.155 | 192 | 664 |
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 |
Pippin Prep Size Select (350-450 bp select):
Run Final Product on qPCR for check
Result from qPCR check: