Procedure

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 3´A-overhangs will gradually be lost during storage.

Note

Based on protocols from Qiagen and Finnzymes.