Procedure pour l'addition de 3' A-overhangs (TA cloning)

Phusion™ DNA Polymerases create blunt end DNA products. When cloning fragments amplified with Phusion DNA Polymerases, blunt end cloning is recommended.

If TA cloning is required, it can be performed by adding 3' A overhangs to the blunt PCR product with a different polymerase (e.g. DyNAzyme™ II DNA Polymerase, Finnzymes).

Procedure for adding 3' A-overhangs (TA cloning)

1. Purify the PCR product (e.g. with a commercial PCR purification kit or phenol extraction and DNA precipitation)
Before adding the overhangs it is very important to remove all the Phusion DNA Polymerase by purifying the PCR product carefully, as the proofreading activity in Phusion DNA Polymerase is very strong. Any remaining Phusion DNA Polymerase will degrade the A overhangs, thus creating blunt ends again.

2. A-addition with DyNAzyme II DNA Polymerase (or Taq DNA polymerase)

Reaction components:

• Purified PCR product
• 0.2 mM dATP
• 1x Optimized DyNAzyme™ Buffer
• 1 U DyNAzyme II DNA Polymerase

Incubate 20 min at 72 °C.

3. Proceed to TA cloning. For optimal ligation efficiency, we recommend using fresh PCR products, since 3´A-overhangs will gradually be lost during storage.