blunt&sticky-1 Ligation?

Mark Henteleff markh at ahi.pbrc.hawaii.edu
Fri Apr 19 19:23:55 EST 1996


I have been trying to ligate a small piece of dsDNA (60bps,
constructed by building overlapping sense and antisense oligos
with restriction site overhangs at each end) into a cDNA with
corresponding restriction sites.  The sites are Nsi (4bp overhang)
and XcmI (1bp overhang).  The small piece has a unique BstEII site
built into it to confirm ligation for subsequent screening.  The
problem is that after screening 50 colonies from a plate that showed
3X over background, there were no positives for the incorporation of
this piece.  According to the NEB catalog, Xcm is a low efficient
ligation site that requires a high concentration of ligase.  I have
found that high ligase inhibits cohesive end ligations.  I have tried
sequential ligations starting in low ligase and moving to high.  I
have also tried Boeringer-Mannheims Rapid DNA ligation kit and several
different molar ratio ligations....all with no success.  My question
is two fold:  a) how can i decrease my background ligations of
re-ligated single cuts without having to go to CIPing the big piece...
an option i would rather not take since my small overlapping oligos
already are without phos. ends. and b) how can i ligate this piece in
given this one end cohesive and one end blunt(or near blunt) problem.

thanks
Mark H.





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