Ligation of PvuI site

Winoto lab lab_winoto at maillink.berkeley.edu.
Mon Jun 27 20:56:50 EST 1994


In article <CrHMqK.LBp at magnus1.com>
maryr at magnus1.com (Bulletin board login) writes:

> I have been trying to set up a plasmid construct using a Pvu I site.  This 
> site does not want to ligate.  I've tried BRL PvuI with no success but, have
> had a little bit of success with PVuI from New ENg. Biolabs but, no success 
> lately even with NEB enzyme.  Any help or suggestions would be greatly 
> appreciated.  Regards, Mary

You may want to try doing your ligations in lowmelt agarose.  We've
noticed that the success of blunt end ligations is greater in LMT
agarose verses solution ligation.  Since PvuI leaves a 3' 2bp overhang
of the AT variety its not likely to anneal as readily as 4bp overhangs.
Alternatively you could follow the ligation conditions for T-vectors
which have only a 1bp overhang (ask Clonetech to FAX you a copy of
their TA Cloning Kit protocols:1-800-955-6288).  If you want to try the
low melt approach here are the conditions:

1.0 ul 1mg/ml BSA
1.0 ul 100mM MgCl2
1.0 ul 100mM ATP
2.0 ul  Buffer 2 from NEB
2.0 ul  50mM spermidine (fresh)
2.0 ul  10mM DTT
9.0 ul insert DNA in LMT agarose (melt at 70C, cool to 50C before
addition)
1.0 ul plasmid DNA (cut and ciped)
1.0 ul T4 DNA ligase (5 units/ul)

ligate overnight at RT or at 16C whatever you perfer (RT works better
for me)
The amount of DNA you will have will vary but I follow these rules of
thumb:
1.  Cut enough insert DNA such that your the yeild of target fragments
is about 2-5 ug

2.  Cut CIP 2-3 ug of plasmid

Run the digests into LMT agarose (use TAE buffer) and isolate in the
smallest possible volume: cut away all of the agarose save for the
piece that has the highest concentation of DNA/mg agarose (estimate by
fluoresence intensity).  Use these prepartions for your ligations. 
Transformation:  melt the ligation mixes at 70C for one or two minutes
dilute 1:5 in ddH2O and transform 5 ul by electroporation or the
classical method.  If you use electorporation and the marker is AmpR on
the plasmid you can plate them immediately on selective plates without
adding LB or SOC and get good results.  



More information about the Methods mailing list