I need your help with a blunt-end cloning and bacterial transformation. I digested the insert with AanI (fermentas) and gel-extracted it using Sigma GenElute kit; to improve Dna quality, the insert was precipitated using NaOAc and Isopropanol over the weekend. Then I checked it on agarose gel. I also digested the acceptor plasmid with SmaI (Fermentas) and dephosphorylated it with Antarctic Phosphatase (NEB).
Ligation was performed using T4 Dna ligase (NEB) in 10 ul final volume: I used the 1:1, 1:3 and 1:6 molar ratios (v:i). I also re-digested the ligation with SmaI (which should not be present anymore in the recombinant plasmid). Then I used the 'Efficiency Library DH5alpha' Kit (Invitrogen) for the transformation, but all the clones screened contain only the empty vector!
I also performed the following controls:
1) tranformation using the acceptor vector digested and self-ligated compared to the same vector digested but not ligated: the former produced lots of colonies while the latter only few: therefore the ligase worked fine.
2) tranformation using the acceptor vector digested, dephosphorylated and self-ligated compared to the same vector digested not dephosphorylated and self-ligated: the former had a lower transformation efficiency, so I can desume the phosphatase worked.
3) tranformation using the acceptor vector digested, dephosphorylated, self-ligated and re-digested compared with the the same vector digested, dephosphorylated, self-ligated and not re-digested: the former had a much lower transformation efficiency (sometimes even no transformants are produced), therefore the second digestion remove almost completely the fraction of plasmid which is not digested during the first round.
I don't know what to do anymore!!!! Please, I'd appreciate any suggestion you could provide me with. thanks
Edited by tonnox82, 09 November 2010 - 07:34 AM.