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 (5 uM working)
- Illpcr1 (5 uM working)
- Pool of above 2 primers (2.5 uM working of each)
...
Normalize to between 20 ng/ul and 150 ng/ul
MseI oligo Annealing
Only needs to be done after first making working stock.
Add 1200 ul std TE to two 2 ml tubes.
Add 150 ul MseI1
Restriction Digestion
(Keep MM and reaction plates on ice)
...
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 2.125 ul of the MM directly to the previous PCR
...