Three fragment ligation

Darren Natale dnatale at box-d.nih.gov
Fri Jan 17 10:44:28 EST 1997


Andrew Doherty wrote:
> 
> Hi everyone
> 
> There must be lots of people out there ligating three bits of DNA
> together instead of just two ...
> My vector is pcDNA1/amp (dephosphroylated) cut with NotI/SphI, my
> receptor cut with NotI/HindIII and GFP cut with HindIII/SphI. So putting
> all three together should give me a circular plasmid. The ligation looks
> fine on a gel - the GFP (800bp) and receptor (3Kb) both disappear and I
> get a ladder of products. But no colonies!! I know that the bugs are
> fine, as other transformations are working well - so my question is, are
> there any tricks in getting this sort of ligation to work or is it just
> luck??!!@# I'm using BM's T4 ligase at 4 deg C overnight.

One possibility is that the concentration of one or both of your inserts
is too high, such that self-ligation is favored.  Since the vector is 
dephosphorylated, you can reverse the usual "more insert than vector"
rule
and put in a molar excess of vector.  The insertion steps will be
disfavored
relative to the circularization step, but this is okay for the three
types 
of ends you are using.  You also do not need to do the ligation at 4
degrees,
which really slows things down.  If you do not want to go as high as
room
temperature, then do the ligation at 16 or so overnight.

Darren Natale
dnatale at box-d.nih.gov



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