Knight: Addition of 3' A-overhangs

Overview

This protocol adds 3' A-overhangs to a PCR product rendering it suitable for TA cloning.

Materials

• Taq polymerase
• A heat block equilibrated to 72°C

Procedure

1. 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.
2. Incubate at 72°C for 8-10 minutes (do not cycle).
3. Place on ice and use immediately in the TOPO cloning reaction.

Notes

• I have not yet tried this protocol!
• An alternative is TdT with ddATP.

References

1. Invitrogen TOPO TA Cloning® Kit for Sequencing manual (page 21)