Use iProof Polymerase for this. We can play around with KAPA afterwards to see if we don’t need 2 taqs in house
Reagents and Ordering
- EcoR1 (20,000 units/ml)
- MseI (10,000 units/ml)
- T4 DNA ligase buffer (400,000 units/ml)
- DNA polymerase (BioRad iProof or KAPA HiFi)
- BSA (1 mg/ml)-We have this in 20 mg/ul
- 1 M NaCl-We have this at 5M
- DMSO
- EcoR1 adaptors (We need 1 uM MIDed in plates)
- Mse1 Adaptors (10 uM stock probably need to dilute from 100 uM)
- IIllpcr2 (10 uM working)
- Illpcr1 (10 uM working)
- Pool of above 2 primers (5 uM working of each)
...
Add 189 ul of low EDTA TE store at 4C for a month or -20C for longer.
PCR Amplification
Pool ligated plates matching well to well in sets of 4 with one set of 2 avoiding combining the same MIDs together.:
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
|---|---|---|---|---|
1 | ||||
2 | ||||
3 | ||||
4 | ||||
5 | ||||
6 |
Add 16 ul of PCR1 MM to each well of plates
...