1CHIP (Chipmunk 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 | 220 | 253 |
5M NaCl | 0.12 | 220 | 26.4 |
1 mg/ml BSA | 0.6 | 220 | 132 |
H2O | 0.73 | 220 | 160.6 |
MseI (enzyme) | 0.12 | 220 | 26.4 |
EcoR1 (enzyme) | 0.28 | 220 | 61.6 |
Total | 3 | 220 | 660 |
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 | 220 | 220 |
H2O | 0.112 | 220 | 24.6 |
10x T4 Buffer | 0.1 | 220 | 22 |
5M NaCl | 0.01 | 220 | 2.2 |
1 mg/ml BSA | 0.05 | 220 | 11 |
T4 DNA ligase | 0.1675 | 220 | 37 |
Total | 1.4 | 220 | 308 |
Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.
CHP1: Eco5
CHP2:Eco6
PCR1:
Reagent | ul/rxn | rxns | ul needed (x1.4) |
|---|---|---|---|
H2O | 9.52 | 110 | 1047.2 |
5x iProof buffer | 4 | 110 | 440 |
10 mM dNTPs | 0.4 | 110 | 44 |
50 mM MgCl2 | 0.4 | 110 | 44 |
5 uM Illumina Primers | 1.33 | 110 | 146.3 |
iProof TAQ | 0.2 | 110 | 22 |
DMSO | 0.15 | 110 | 16.5 |
total | 16 | 110 | 1760 |
Add 16 ul of PCR1 MM to 80 wells of a new hard shell PCR plate 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 | 19X | 0:30 |
60 | 19X | 0:30 |
72 | 19X | 0:40 |
72 | 1X | 10:00 |
4* | 1X* | 0:00* |
*pause step