Oligo length check

Tomi Makela tomakela at cc.helsinki.fi
Tue Nov 26 12:03:43 EST 1991


In article <1991Nov26.042540.19587 at usenet.ins.cwru.edu>, djt2 at po.CWRU.Edu (Dennis J. Templeton) writes:
> 
> 
In a previous article, msalminen at nphi.fi () says:
> 
> >Question:
> >
> >If i run a 20%, (95% akrylamide/5% bis) urea denaturing sequencing gel loading
> >500-1000 ng oligo, will i detect one base length differences after ethidium
> >bromide staining, or will the base composition affect migration too much? I run
> >the gel in a BioRad miniprotean device (about 10 cm cel). My oligos are mostly
> >20-22 mers with a couple of 16 and 25-mers.
> >
djt2 at po.CWRV.Edu (Dennis Templeton) answers that this is a bad idea because:
> 
> Reason 1:
> 
> The oligo is liable to soak out in the ethidium stain, leading to loss and 
> cross contamination

Not true; we routinely analyze all the oligos we make by running them 
in a 20 % gel and afterwards staining with ethidum bromide. Soaking 
is not a problem, and staining is a good way of checking the amount of 
non-full-length oligo. We have analysed over 600 oligos this way, and 
we have also noticed an interesting point, which is that using non-
denaturing gels the secondary structures in the oligos are conveniently
detected. 

> Note 2:  Are you sure you need to purify the oligo?  why?  most
> applications work with "crude" oligos.  Try it out, maybe?

A good idea! 

Tomi Makela
Dept. of Virology
University of Helsinki



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