...
Add 3 ul Digestion MM to 3 plates using the 8 channel
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
Reagent | ul/rxn | rxns | ul needed (1.5x) |
10x T4 Buffer | 1.15 | 480400 | 690460 |
5M NaCl | 0.12 | 480400 | 7248 |
1 mg/ml BSA | 0.6 | 480400 | 360240 |
H2O | 0.73 | 480400 | 438292 |
MseI (enzyme) | 0.12 | 480400 | 7248 |
EcoR1 (enzyme) | 0.28 | 480400 | 168112 |
Total | 3 | 480 | 1800 |
Tubes:
400 | 1200 |
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