Knight: Addition of 3' A-overhangs
This protocol adds 3' A-overhangs to a PCR product rendering it suitable for TA cloning.
- Taq polymerase
- A heat block equilibrated to 72°C
- After amplification with the proofreading polymerase, place vials on ice and add 0.7-1 unit of Taq polymerase per tube. Mix well. It is not necessary to change the buffer. A sufficient number of PCR products will retain the 3´ A-overhangs.
- Incubate at 72°C for 8-10 minutes (do not cycle).
- Place on ice and use immediately in the TOPO cloning reaction.
- I have not yet tried this protocol!
- An alternative is TdT with ddATP.