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Adding 3' A overhang to a PCR product

Author: S. Mohsen Hosseini, Hospital for Sick Children, To
Source: Protocol Online
Date Added: Mon Feb 02 2009
Date Modified: Mon Feb 02 2009
Abstract: Protocol for adding 3' A overhang to PCR products for TA cloning.



  1. Purify the PCR product. Before adding the overhangs it is very important to remove all the Proofreading DNA Polymerase (Pfu) by purifying the PCR product carefully (e.g. with a commercial PCR purification kit or phenol extraction and DNA precipitation); since the proofreading activity of DNA Polymerase will degrade the A overhangs, creating blunt ends again.
  2. Prepare Taq DNA polymerase reaction mix for a typical 20 - 50 μl reaction:
       Final Concentration    Vol (μl)
    Purified PCR product    0.15 to 1.5 pmol    Varies*
    dATP (10 mM)    0.2 mM    1
    PCR Buffer with Mg (10x)    1x (1.5 mM MgCl2)    5
    Taq DNA Polymerase (5 U/μl)    1U    0.2
    ddH2O       to 50 μl

* The A-addition reaction works best when a specific amount of the PCR product is used. The recommended amount is 10–100 ng PCR product for each 100 bp length of the PCR product. This corresponds to 0.15–1.5 pmol PCR product (see table below).

PCR product size    Amount of PCR product to use
100 bp   10–100 ng
250 bp   25–250 ng
1000 bp   100–1000 ng
  1. Incubate 20 min at 72 °C.
  2. Proceed to TA cloning. For optimal ligation efficiency, it's best to use fresh PCR products, since 3ŽA-overhangs will gradually be lost during storage.


Based on protocols from Qiagen and Finnzymes.

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