1TORRES
Restriction Digestion
(Keep MM and reaction plates on ice)
Set Incubator to 37 C
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 | 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 |
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.
Adaptor Ligation
Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.
Reagent | ul/rxn | rxns | ul needed (1.6x) |
|---|---|---|---|
MseI oligo | 1 | 450 | 450 |
H2O | 0.112 | 450 | 50.4 |
10x T4 Buffer | 0.1 | 450 | 45 |
5M NaCl | 0.01 | 450 | 4.5 |
1 mg/ml BSA | 0.05 | 450 | 22.5 |
T4 DNA ligase | 0.1675 | 450 | 75.4 |
Total | 1.4 | 450 | 630 |
Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.
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 16 ul of PCR1 MM to each well of 2+12(for 4Trout) new hard shell PCR plates with AssistPlus program 16ulMMDisp on 125ul ViaFlo
Add 4uL of template from pooled plates with Benchsmart or Assist Plus PoolWINPlatePCR on 12.5 ul ViaFlo
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 2.155 ul of the MM directly to the previous PCR
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 |
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