blunt ending the vector followed by the ligation - (Jun/21/2007 )

Dear All,
I have a plasmid vector having a cohesive ends which is 4 kb gel purified after cutting with ECO- RI (2 ECO- RI Sites).
I have the PCR product:PCR carried out with pfu DNA poymerase so obiviously blunt ends of the PCR product will be present.
Now I have to treat the vector with the klenow for the fill-in reaction.
Will klenow add the phosphate group at 3' end of the vecor or no? Do I need to treat with polynucleotide kinase for phosphorylation?
I have a question in my mind that do I need to phosphorylate the vector or PCR product to increase the efficiency of ligation?
Can anybody let me know this?
Vineet

---

Yes.


No



Depends on personal preference.
1) If you dephosphorylate your vector, you need to phosphorylate the PCR insert (unless your primers were specifically purchased 5' phosphorylated.) However phosphorylating the insert results in concatermer formation. You need to screen against such events. Main problem is that this method requires 2 additional steps. The more steps you need to do the more things that can go wrong. However you need only to screen fewer colonies to get the right clone.

2) However if you leave the vector as it is, the vector can religate to itself. This result in high levels of background. However as the insert ends are free of phosphorylation, no concatemers can form. This thus requires screening alot more colonies. But as there are fewer steps there are fewer things to go wrong.

---