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)
...
Add 1200 ul std TE to two 2 ml tubes.
Add 150 ul MseI1 to each tube
Add 150 ul Mse2 to each tube
Close, vortex, and spin down both tubes.
Heat to 95C for 5 minutes and allow to slowly cool to RT. (This step will also need to be done for the 8th EcoRI adaptor if it is available.)
Restriction Digestion
(Keep MM and reaction plates on ice)
...
Size Selection
Blue Pippin?
Gel Extraction?