4 replies to this topic

[arwen76]

Hi everybody,

does anybody know if it is necessary to phosphorylate a pcr product (generated by pfu polymerase so it should have blunt ens if I'm right) before ligating it into a plasmid with blunt ends?( plasmid cut with KpnI then generated blunt end with t4 dna polymerase).

It would be very, very helpful to get an answer. Nobody in my lab seems to be qualified....

Thanks

[han526]

Hi There:

You do need to phosphorylate the PCR product and de-phosphorylate your vector. I am a pro.
Stanley

[Janina]

Hi Arwen!

Did your blunt end ligation work? I am asking because I have several problems with my cloning... My Insert seems to be ligated (controlled by Colony-PCR) but then my blunt end site does not work anymore... So perhaps, it is a general problem with blunt end ligation...  

[arwen76]

Since my question I didn't try the ligation again. But I also found out that phosphorylation of the pcr product (if the fw primer is not phosphorylated) is necessary. Thank for replying...

I know that my blunt end site will not work anymore because there is no restriction site. I created the site by using t4 dna polymerase. In my case I blunted 3' protruding termini resulting from restriction with KpnI. So the restricion site is not there anymore. How do you create the blunt end (is it a restriction site like e.g. EcoRV?).

When mine has worked I'll report it.

Good luck to everbody.

Arwen...

[Janina]

In my case, it is a restriction site. It is BalI/MscI/MlsI ... But now, we seem to be close to the solution of our problem... Some little experiments left to do...