Blunt ended ligation

[Ron Kagan]

In article <3dqfi4$6jl at nermal.cs.uoguelph.ca> Shayan Sharif,
ssharif at uoguelph.ca writes:
>I have been trying to do a blunt-ended ligation, but it doesn't seem to 
>be working. At first I checked the transformation step. With the intact 
>pGEM3 it works just fine, but I cannot get any colony (either with or 
>without the insert) with the ligated vector. Moreover, agarose gel 
>analysis, using supercoiled marker, shows that the DNA in ligation 
>mixture is still in linear form.
>I have tried these to sort out the problem:
>
>1- Different molar ratios of insert:vector from 3:1 to 1:3.
>2- Religating the vector without any insert.
>3- Different incubation temperatures and lengths of time.
>4- Comparing T4 DNA ligases supplied by NEB, Pharmacia, Gibco, and
Promega
>(approxiamtely 5-6 Weiss units or equivalent amount of NEB T4 ligase).
>5- A range of MgCl2 and ATP final concentrations, from 50 to 100 mM
>and 0.1 to 1 mM respctively.
>
>Any suggestion is welcome. Thanks.

I do my blunt end ligations with the T4 ligase from Gibco-BRL (1 unit)
and the 5X buffer supplied by Gibco.  I find that 4C overnight works best
for me.  I do my ligations in a final volume of 15 microliter.  Just
before the transformation, I dilute the ligation mix 1:5 with water or TE
and use 3 microliter per transformation.  The dilution supposedly
minimizes the effect of certain inhibitors of transformation in the
ligation mix.  

Ron Kagan

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"You cannot strengthen one by weakening another; and you cannot
 add to the stature of a dwarf by cutting the leg off a giant."

                                - Benjamin Franklin (1706-1790)

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Ron Kagan
rkagan at ewald.mbi.ucla.edu