...
Reagent | ul/rxn | rxns | ul needed (1.5x) |
10x T4 Buffer | 1.15 | 400 | 460 |
5M NaCl | 0.12 | 400 | 48 |
1 mg/ml BSA | 0.6 | 400 | 240 |
H2O | 0.73 | 400 | 292 |
MseI (enzyme) | 0.12 | 400 | 48 |
EcoR1 (enzyme) | 0.28 | 400 | 112 |
Total | 3 | 400 | 1200 |
Tubes:
...
Reagent
...
ul/rxn
...
rxns
...
ul needed (1.5x)
...
10x T4 Buffer
...
1.15
...
56
...
69
...
5M NaCl
...
0.12
...
56
...
7.2
...
1 mg/ml BSA
...
0.6
...
56
...
36
...
H2O
...
0.73
...
56
...
43.8
...
MseI (enzyme)
...
0.12
...
56
...
7.2
...
EcoR1 (enzyme)
...
0.28
...
56
...
16.8
...
Total
...
3
...
56
...
180
Add 6 ul template to each plate with Benchsmart. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 65C in EcoBGC oven for 1 hour.
...
Reagent | ul/rxn | rxns | ul needed (1.6x) |
|---|---|---|---|
MseI oligo | 1 | 536450 | 661450 |
H2O | 0.112 | 53645074 | 50.4 |
10x T4 Buffer | 0.1 | 536450 | 66.145 |
5M NaCl | 0.01 | 536450 | 64.615 |
1 mg/ml BSA | 0.05 | 53645033 | 22.5 |
T4 DNA ligase | 0.1675 | 536450 | 7275.74 |
Total | 1.4 | 536450 | 110.72 630 |
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