1ABAX (WY Toad Project)
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 | 480 | 690 |
5M NaCl | 0.12 | 480 | 72 |
1 mg/ml BSA | 0.6 | 480 | 360 |
H2O | 0.73 | 480 | 438 |
MseI (enzyme) | 0.12 | 480 | 72 |
EcoR1 (enzyme) | 0.28 | 480 | 168 |
Total | 3 | 480 | Â 1800 |
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 | Â 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.
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 | 536 | 661 |
H2O | 0.112 | 536 | 74 |
10x T4 Buffer | 0.1 | 536 | 66.1 |
5M NaCl | 0.01 | 536 | 6.61 |
1 mg/ml BSA | 0.05 | 536 | 33 |
T4 DNA ligase | 0.1675 | 536 | 72.7 |
Total | 1.4 | 536 | Â 110.72 |
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 | 268 | 2761 |
5x iProof buffer | 4 | 268 | 1160 |
10 mM dNTPs | 0.4 | 268 | 116 |
50 mM MgCl2 | 0.4 | 268 | 116 |
5 uM Illumina Primers | 1.33 | 268 | 385 |
iProof TAQ | 0.2 | 268 | 58 |
DMSO | 0.15 | 268 | 43.5 |
total | 16 | 268 | 4640 |
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 2.155 ul of the MM directly to the previous PCR
Reagent | ul/rxn | rxns | ul needed (x 1.6) |
5x Iproof buffer | 0.425 | 272 | 136 |
10 mM dNTPs | 0.4 | 272 | 128 |
Primers | 1.33 | 272 | 426 |
Total | 2.155 | 272 | 690 |
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 |
Size Select at 275-375bp
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