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)
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Seal, vortex, and spin tubes. You will need 2 tubes for this prep.
MseI oligo Annealing
Only needs to be done after first making working stock.
Add 1200 ul std TE to two 3 three 2 ml tubes.
Add 150 ul MseI1 to each tube
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