Dephosphorylation nick problem - (May/26/2006 )

Hi all,

I am about to perform a desphorylation of vector before cloning an insert into it. I have never done this before but have heard that doing so is very time efficient. However, I have doubts about what happens to the two nicks that will be in the recommbinant DNA. Does anyone know ?

Kushan

---

where these nicks will come from!!
i think that just discontinuties will form & u can use a ligase to fill them..

---

Okay.. So vector has OH on 3' end but no phosphate on 5' end.. insert has phosphate on 5' ends and OH on 3' ends.. Ligase can only synthesize a phosphodiester bond if it has a 5' phosphate and 3' OH. So it will synthesize phosphodiester bond between the 3' OH of the vector and 5' phosphate of the insert on one strand and the same kind of bond on the opposite strand.. But when the 3' OH of the insert meets with the 5' end of the vector (where the phosphate used to be before dephosphorylation) there is no phosphate and hence no phosphodiester bond. This is how you will have two nicks on the opposite strands. And ligate cannot fix it until there is 5' phosphate there..


does anyone has an idea what will happen to this nick when used to transform E coli..

---