New oligo bridges arrived. Trying a ligase cycling reaction that will include the pJET1.2 plasmid into the product.
Mostly taken from here. Only major differences:
- Prepared diluted oligo bridge stocks, as the previous stocks I was using were 10x more concentrated than called for in the protocol.
- pJET1.2 plasmid is at 50 ng/µL, which is slightly below 30 nM concentration. Will be using straight plasmid for this reaction.
- Using a 20 µL reaction volume for the PNK reaction.
- Using a 45 µL reaction volume for the LCR reaction (will do this on Monday).
| Reagent || Volume
| Clean PCR or dsDNA || 5 parts, 2 µL per part
| 10x T4 Ligation buf (NEB) || 2.0
| T4 PNK (NEB) || 0.5
| dH2O || 7.5 μL
| || 20.0 μL
- Incubate at 37°C/ 30 min.
- Heat-inactivate PNK at 65°C/ 20 min.