...
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.
...