sequenase (getting around the glycerol)

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Tue Jan 10 11:44:44 EST 1995


P. Vescio wrote:

> [Concerning the glycerol gel effect originating from the sequenase
>  storage buffer]

> --- Which method has given the best success?  Diluting the enzyme into the
> glycerol buffer once and running reactions on glycerol tolerant gels  or
> Diluting the enzyme 1:8 before each use.

We found that the glycerol effect can be mostly eliminated by flushing the
wells just after the dyes have run in, so this has ceased to be an issue
for us.  The effect is caused by an interaction between the glycerol
and the borate in the buffer which generates a charged species that
runs into the gel and creates a zone of distorted migration.  Apparently
it takes a while for this species to form in the well, during which time
the glycerol of course isn't going anywhere.  So you have a chance to
just physically remove it.

If your ladders have to be perfect, then the glycerol tolerant buffer
is probably the thing to do.  Note: it's the buffer, not the gel 
matrix that's involved in this problem.

I never understood what the dilution was supposed to accomplish.
We just add the required number
of units direct from the concentrated tube to the cocktail and get on 
with it.  So I guess that amounts to diluting into reaction buffer.

Steve Hardies, Dept. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu




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