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)
...
Pool plates matching well to well in sets of 3 or 4 avoiding combining the same MIDs together.
...
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
H2O | 9.52 | 777650 | 7397.056188 |
5x iProof buffer | 4 | 777650 | 31082600 |
10 mM dNTPs | 0.4 | 777650 | 310.8260 |
50 mM MgCl2 | 0.4 | 777650 | 310.8260 |
2.5 uM Illumina Primers | 1.33 | 777650 | 1033864.45 |
iProof TAQ | 0.2 | 777650 | 155.4130 |
DMSO | 0.15 | 777650 | 11697.555 |
total | 16 | 777650 | 1243210400 |
Add 4 ul of restriction/ligation products to each well
...
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
5x Iproof buffer | 0.425 | 777650 | 330276.22525 |
10 mM dNTPs | 0.4 | 777650 | 310.8260 |
Primers | 1.33 | 777650 | 1033864.415 |
Total | 2.155 | 777650 | 16741400.43575 |
Then continue with the last four unlisted steps for GBS1 from above:
...