My vector is about 7Kb. The cloning site is EcoRI-HindIII, but later I found there is another EcoRI site 1Kb downstream of the vector. Good thing there is a NheI site, 70bp upstream of EcoRI-HindIII site.
I've done the following,
1)Restriction digest the vector with NheI and HindIII, DNA gel purified and drop dialyzed in water.
2)PCR my insert (350bp), restriction digested with EcoRI and HindIII, gel purified and drop dialyzed.
3)PCR, restriction cut and gel purified the 70bp NheI and EcoRI fragment. This fragment is essential for expression.
The ideal ligated product is NheI-----------EcoRI------------HindIII.
Then I tried DNA ligation using Invitrogen T4 DNA ligase with vector (65fmol), insert (165fmol) and fragment (165fmol). But so far no colonies yet (DH5alpha chemical transformed, Invitrogen). Please let me know if anyone has any suggestion?
Edited by bispecific antibody, 12 September 2012 - 08:23 AM.