Ligation failed

newsnet customer customer at newsnet.com
Sat May 6 09:19:09 EST 2006


"Austin P. So (Hae Jin)"
> newsnet customer wrote:
> > Hi,
> >
> > I have transformed an insert/gene (2kb) into a cloning vector.
> > Then cut the gene out to ensure it has sticky ends.
> > Also, I have linearised an expression vector (11kb) with compatible
> > sticky ends to the insert.
> >
> > Here is the ligation reaction:
> >
> > insert (12ng/ul)                     2ul
> > linearised vector (6ng/ul)        4ul
> > 10X lig. buffer                       1ul
> > T4 ligase                              1ul
> > Water                                  2ul
> > TOTAL                                 10ul
> >
> > overnight incubation @ 4C in accordance to Roche T4 ligase information
> > sheet.
> > Total DNA in ligation reaction is 48ng.
> > insert:vector ratio is 1:1 in accordance to Roche T4 ligase information
> > sheet when insert to vector size are significantly different.
>
> Actually, no, the insert to vector ratio is ~1:5...unless you are
> cutting asymmetrically, then you don't have enough. You have to go by
> molar ratio (or the equivalent of dividing mass by size), not mass ratio.


That ratio doesn't look right to me. And please define molar ratio for DNA?


> The other thing is that you should check if the Roche buffer has PEG
> (see below).
>
> > 1. chemically competent cells are not suitable for vector >10kb - HIGHLY
> > PROBABLE
>
> This is easy enough to check...just do a control transformation with a
> no insert ligation reaction. If it can handle 11 kb, then it can handle
> an extra 2 kb.


Fair enough.


> > 3. Total DNA concentration is too high to promote circularization of
> > vector - LOWLY PROBABLE AS STILL EXPECT SOME COLONIES
>
> Your concentration of DNA is on the low side, and that increases the
> chance of circularization. Both the insert and the vector are large
> enough that the intramolecular ligation has a higher chance than the
> intermolecular (did I get the terms mixed up ? :/), so there is a large
> chance that both your insert and your vector could have independently
> circularized.


You actually want your total DNA on the low side to promote circularization
otherwise you will never get ligation. In otherwords, two strands of linear
DNA will never ligate even if they had compatible ends. Sambrook even
suggest using vector/insert at concentrations as low as 10ng.

Cheers,
ST





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