Top : Molecular Biology : Molecular Cloning : TA Cloning : Addition of 3'-A Overhangs (A-Tailing) to PCR Product

Addition of 3'-A Overhangs (A-Tailing) to PCR Product

Source: Protocol Online
Date Added: Mon Feb 02 2009
Date Modified: Mon Feb 02 2009
Abstract: PCR product amplified by proofreading polymerases is blunt-ended because proofreading polymerases possess 3→5 exonuclease activity that removes the 3-A overhangs necessary for TA cloning. However, 3-A overhangs can be added to blunt-end fragments using a Taq polymerase after PCR amplification.

  1. After amplification with a proofreading polymerase, place samples on ice and add 0.7-1 unit of Taq polymerase per tube directly into the PCR reaction tube. Mix well.
  2. Incubate at 72C for 8-10 minutes.
  3. Place the reaction on ice and use it immediately for ligation reaction with a TA cloning vector such as the TOPO TA cloning vector from Invitrogen.
  4. Alternatively, the PCR product can be purified before used in ligation reaction as follows.
  5. Extract reaction immediately with an equal volume of phenol-chloroform to remove all of the polymerases.
  6. Precipitate the DNA by adding 1/10 volume of 3 M sodium acetate and 2X volume of 100% ethanol.
  7. Centrifuge at maximum speed (14,000 rpm) for 5 minutes at room temperature to pellet the DNA.
  8. Remove the ethanol, rinse the pellet with 80% ethanol, and allow to air dry.
  9. Resuspend the pellet in TE buffer to the starting volume of the PCR amplification reaction. The PCR amplification product is now ready
    for ligation into a TA vector.
     
Printer friendly page