Knight: Addition of 3' A-overhangs

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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


  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.


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


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