Adding 3A overhang to a PCR product

 

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	Varieundefined
   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

~undefined 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.