Digestion and ligation - Do I omit the dephosphorylation steps? (Sep/02/2008 )

Dear all,

If cutting with two different REs produces ends which may not be complementray, thus no self-ligation, does that mean I could omit the dephosphorylation of the vector?

Speaking of dephosphorylation and phosphorylation, do I have to do any of these two on my PCR-amplified fragments? Wouldn't normal polymerization produce a 5' end with the phosphate?

I'm really new in all these molecular techniques.

Thanks a lot for the help.

When you prepare your vector with a double digestion (no self ligation possible) you don't need to dephosphorylate your vector.

Regards

Oh, thanks a lot!

The main point of using two REs is to determine the direction of cloning, not stop self-ligation. If one of the digests only works 95%, you're much more likely to re-ligate the 5% single-cut vector than ligate your insert into the 95% double-cut vector.

After much pain using an enzyme that we had become committed to using but that doesn't religate well (long story, please don't get me started), I found that CIP treating the vector after the double digestion stage, plus doing a kill digest after the ligation, worked best. (If you've not come across it, kill-digestion involves adding an RE that is present in the vector backbone, but absent in the recombinant vector. This linearises any / most religated vector.) If you can prove the vector is 100% double-digested, say, by a ligation test that produces a ladder of vector multiples or else by gel purification, you don't need to CIP-treat.

If you CIP-treat the vector, you will need to PNK-treat the PCR product, because most oligos are synthesised without the phosphate group. Remember that the 5' end of the PCR product is your primer, so, no, you don't get a 5' phosphate group.