Does the PCR product amplified using pfu polymerase have 5' phosphate groups to use for blunt cloning?
Hello Emilia,
No, DNA polymerases do not add 5' phosphate groups to PCR products. That's the job of kinases + ATP. You could phosphorylate either your primers before performing the PCR or the final product after completing the synthesis. But for blunt cloning, you may even not need to bother, provided that you deal with a vector that has been cut with a restriction enzyme, because the latter will leave a 5' phosphate on either site of the cut. Thus, ligase will be able to ligate your insert on one strand on either side of the cut. The outcome will be a recircularized plasmid with two nicks on either side of the insert, which will be fixed in vivo by the host after transformation. I have done this many times. This will only work with a vector that hasn't been dephosphorylated, of course. Not phosphorylating your insert will also prevent the formation and cloning of concatemers.
I h ope this is useful
Pierre
- Badges
- Science method