Ligation problems on agarose-isolated fragments

Colin Rasmussen colin at fungus.biochem.ualberta.ca
Wed Feb 28 22:49:05 EST 1996


Mr SMNN Faruque wrote:

> >
> > Marieke
> 
> The SeaPlaque booklet states that in-gel reactions (eg ligation) require that the
> gel buffer has only one tenth the normal amount of EDTA.
> Do you do this or do you find there to be no problem?

I never read the SeaPlaque book (don't much like instruction manuals) but here's how 
ligations are presently being done in my lab.

Boehringer Rapid Ligation Kit.

In tube sitting in a 37°C water bath, mix 2ul DNA dilution buffer with up to 8ul DNA 
in agarose (we usually use 4ul of gel slices containing vector and insert 
respectively).

Add the 10ul of ligation buffer.

Add 1ul of T4 ligase.

Incubate at room temp.

Transform into bugs.

It works.


Colin



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