Q: Double digestion of adjacent restriction sites ?

[Sasha Kraev]

The problem of cloning into two adjacent sites can be overcome by the 
following trick ( unfortunately not an entirely general one ):

1. Cut with a restriction enzyme and dephosphorylate the DNA.

2. Ligate vector and fragment at a concentration of about 1-5 ug /ml

3. Heat inactivate the ligase and dilute 3 to 5 fold with a second
buffer, containing a second enzyme. Digest and subsequently inactivate 
the second enzyme.

4. Add DTT and ATP to reconstitute the ligase buffer. Add ligase and
ligate second time at DNA concentration <1 ug/ ml.
The idea behind this is that you first ligate your fragment onto both 
ends of the vector, so that a second site is now away from the ends,
then cut the molecule into two parts, of which only one, containing the 
origin of replication, survives on transformation, to form the construct 
you need. The second ligation must be performed at a low DNA 
concentration to favor self-ligation over intermolecular ligation.
The trick needs a little bit of planning, such as finding out if
your buffers are compatible or need to be adjusted. Ligase, in fact,
works in a variety of buffers, provided ATP is present at 0.1 mM or 
more. Heat inactivation also requires  re-addition of DTT. When
heat inactivation is not possible,  DNA has to be purified away from
the enzyme ( e.g. by phenol extraction and ethanol precipitation ).

I wonder if this idea has ever been published, but it works all the 
time.
Cheers,

-------------- next part --------------
Alexander Kraev, PhD                     Internet:    kraev at bc.biol.ethz.ch
> Lab.of Biochemistry III                            Phone:      0041-1-632-31-47
> Swiss Federal Inst. Of Technology      FAX:           0041-1-632-12-13
> Universitaetsstr. 16                      URL: http://129.132.39.25/kraev.html/
> CH-8092 Zurich