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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 (2.5 uM working)
- Illpcr1 (5 uM working)
- Pool of above 2 primers (2.5 uM working of each)
Original SOP
Normalize templates
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Reagent | ul/rxn | rxns | ul needed | ||
|---|---|---|---|---|---|
H2O | 9.52 | 1536 (or 768 if not duplicating) | 14622.72 | 5x KAPA HIFI 777 | 7397.05 |
5x iProof buffer | 4 | 1536777 | 61443108 | ||
10 mM dNTPs | 0.4 | 1536777 | 614310.48 | ||
50 mM MgCl2 | 0.4 | 1536777 | 614310.48 | ||
2.5 uM Illumina Primers | 1.33 | 1536777 | 20421033.884 | ||
KAPA HIFI iProof TAQ | 0.2 | 1536777 | 307155.24 | ||
DMSO | 0.15 | 1536777 | 230116.455 | ||
total | 16 | 1536777 | 24806.412432 |
Add 4 ul of restriction/ligation products to each well
Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 30X | 0:30 |
60 | 30X | 0:30 |
72 | 30X | 0:40 |
72 | 1X | 10:00 |
4* | 1X* | 0:00* |
*pause step
Extra PCR?
Add 2.125 ul of the MM directly to the previous PCR
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
5x Iproof buffer | 0.425 | 777 | 330.225 |
10 mM dNTPs | 0.4 | 777 | 310.8 |
Primers | 1.33 | 777 | 1033.41 |
Total | 2.155 | 777 | 1674.435 |
Then continue with the last four unlisted steps for GBS1 from above:
Temp C | Cycles | Time |
|---|---|---|
98 | 1X | 3:00 |
60 | 1X | 2:00 |
72 | 1X | 10:00 |
4 | 1X | 0:00 |
Size Selection
Blue Pippin?