help with ligation

Mr. Chris Hoyle bmbckh at biovax.leeds.ac.uk
Thu Apr 11 14:20:28 EST 1996


In article <AlP0aey00iWk0_gGJv at andrew.cmu.edu>
Victor Yankiwski <vy05+ at andrew.cmu.edu> writes:

> Hi,
> 
> I'm trying to ligate a variety of inserts into pCDNA3 but have not had
> much luck.  The ligations are with cohesive ends, and single cuts were
> treated with calf intestinal alkaline phosphatase.  On my last attempt,
> I used DNA in TE at a molar ratio of 3:1 insert DNA to vector.  This
> time I'm going to try a molar ratio of 10:1 insert to DNA but dont know
> if I should resuspend the DNA in water before proceeding with the
> ligation reaction (carried out at 25C for 1h).  Does TE inhibit the
> activity of T4 ligase?
> 
> Any suggestions regarding ratios and reaction conditions would be appreciated.
> 
> Thanks
> Vic
> vy05 at andrew.cmu.edu


Try resuspending DNA destined for ligation in autoclaved sterile H2O.
If still not working check out on AGE. If still having problems fo not
plate ouT, instead inoclulate directly to selective broth.
Good luck.

------------------------------------------------------
Chris Hoyle

   Department of Biochemistry and Molecular Biology
University of Leeds             phone +44 0113 2333172
Leeds LS2 9JT                   FAX   +44 0113 2333167
Great Britain           e-mail BMBCKH at biovax.leeds.ac.uk
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