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)
...
Normalize to between 20 ng/ul and 150 ng/ul
Illumina Primer Pooling
Add 900 ul std TE to 5 tubes
Add 50 ul of both Illpcr1 and Illpcr2 to each tube
Seal, vortex, and spin tubes. You will need 2 for this prep.
MseI oligo Annealing
Only needs to be done after first making working stock.
Add 1200 ul std TE to two 2 3 ml tubes.
Add 150 ul MseI1 to each tube
...
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
H2O | 9.52 | 700 | 6664 |
5x iProof buffer | 4 | 700 | 2800 |
10 mM dNTPs | 0.4 | 700 | 280 |
50 mM MgCl2 | 0.4 | 700 | 280 |
2.5 uM Illumina Primers | 1.33 | 700 | 931 |
iProof TAQ | 0.2 | 700 | 140 |
DMSO | 0.15 | 700 | 105 |
total | 16 | 700 | 10400 |
...