A-tailing of old PCR products for TA cloning

Sometimes older PCR products need to be cloned by TA-cloning. The A-overhangs will tend to be labile and cloning efficiency can be low. The simplest way to deal with this is to add some of the PCR product to some PCR master mix and run one PCR cycle, with a 15 minute extension step without primers. The Taq-polymerase in the master mix will add A-overhangs to the double stranded pieces of DNA.

I typically do this using Invitrogen PCR Supermix, but any PCR master mix will do just fine. Using Supermix I proceed with the following protocol:

To 100 ul of Supermix add 1 ul of Taq polymerase (Promega). (I like loading some extra Taq into the reaction; just my personal bias. It should work just fine with the Taq in the Supermix, but Invitrogen really skimps on the taq in this mastermix.)

Add 2 ul of PCR product to 4 ul of the Supermix with extra Taq.

Place tube in thermocycler and run the following cycling program:

• 3 min @ 95 deg C
• 1 min @ 50 deg C
• 30 min @ 72 deg C
• hold @ 4 deg C

Proceed to TA cloning without cleanup.