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)
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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.).
EcoRI plate 4 oligo Annealing
SpeedVac plates to dry oligos
Resuspend plate 4a and 4b to 25 uM. We will have to calculate based off initial expected volume and concentration.
Combine 4 ul from each plate in a well to well fashion with 92 ul of TE to create a 1 uM working plate.
Seal, Vortex, and Spin down resulting plate.
Heat to 95C for 5 minutes and allow to slowly cool to RT
Restriction Digestion
(Keep MM and reaction plates on ice)
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