Finnzymes' DNA polymerases and cloning
Phusion® DNA polymerases have an extremely low error rate, which makes them the ideal choice for cloning. Therefore we strongly recommend Phusion DNA polymerases for all cloning applications. Below you can find information about cloning PCR products amplified with Phusion.
However, if you are cloning PCR products amplified with other Finnzymes’ DNA polymerases, you need to consider that different DNA polymerases create different type of ends in the PCR products, which affects the subsequent cloning procedure. Additional information can be found below.
Phusion DNA polymerases – the best choice for high-fidelity cloning
Phusion® DNA Polymerases create blunt end DNA products. When cloning
fragments amplified with Phusion DNA Polymerases, blunt end cloning is
Procedure for adding 3' A-overhangs (TA
1. Purify the PCR product (e.g. with a
commercial PCR purification kit or phenol extraction and DNA
2. A-addition with DyNAzyme II DNA Polymerase (or Taq 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.
Phire Hot Start DNA Polymerase
Phire Hot Start DNA Polymerase creates blunt end PCR products. If you are
cloning fragments amplified with Phire Hot Start DNA Polymerase, blunt end
cloning is recommended.
DyNAzyme EXT DNA Polymerase
DyNAzyme EXT can create both blunt ends and A-overhangs to the PCR products. Products amplified with DyNAzyme EXT can be cloned in both blunt and TA vectors. Blunt ends and A-overhangs always coexist in the product, but in certain conditions one of them may be more dominant. Some generalization about this can be made as follows:
DyNAzyme II and DyNAzyme I DNA Polymerases
Both DyNAzyme II and DyNAzyme I create A-overhangs in the PCR products. If
PCR products amplified with DyNAzyme I or II need to be cloned, TA cloning can