Addition of 3'-A Overhangs (A-Tailing) to PCR Product
|Date Added:||Mon Jan 02 2006|
|Date Modified:||Mon Jan 02 2006|
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.
- 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.
- Incubate at 72°C for 8-10 minutes.
- 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.
- Alternatively, the PCR product can be purified before used in ligation
reaction as follows.
- Extract reaction immediately with an equal volume of phenol-chloroform to
remove all of the polymerases.
- Precipitate the DNA by adding 1/10 volume of 3 M sodium acetate and 2X
volume of 100% ethanol.
- Centrifuge at maximum speed (14,000 rpm) for 5 minutes at room temperature
to pellet the DNA.
- Remove the ethanol, rinse the pellet with 80% ethanol, and allow to air
- 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.