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 (2.5 uM working)
Original SOP
Restriction Digestion
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
10x T4 Buffer | 1.15 | 1700 | 1955 |
5M NaCl | 0.12 | 1700 | 204 |
1 mg/ml BSA | 0.6 | 1700 | 1020 |
H2O | 0.73 | 1700 | 1241 |
MseI (enzyme) | 0.12 | 1700 | 204 |
EcoR1 (enzyme) | 0.28 | 1700 | 476 |
Total | 3 | 1700 | 5100 |
Adaptor Ligation
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
MseI oligo | 1 | 1700 | 1700 |
H2O | 0.112 | 1700 | 190.4 |
10x T4 Buffer | 0.1 | 1700 | 170 |
5M NaCl | 0.01 | 1700 | 17 |
1 mg/ml BSA | 0.05 | 1700 | 85 |
T4 DNA ligase | 0.1675 | 1700 | 284.75 |
Total | 1.4(395) | 1700 | 2447.15 |
PCR Amplification
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
H2O | 9.67 | ||
5x Iproof buffer | 4 | ||
10 mM dNTPs | 0.4 | ||
50 mM MgCl2 | 0.4 | ||
2.5 uM Illumina Primers | 1.33 | ||
Proof Taq | 0.2 | ||
DMSO | 0.15 |
Add 4 ul of restriction/ligation products to each well
Extra PCR?
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
5x Iproof buffer | 0.425 | ||
10 mM dNTPs | 0.4 | ||
Primers | 1.33 |