Oligo length check

Dennis J. Templeton djt2 at po.CWRU.Edu
Mon Nov 25 23:25:40 EST 1991


In a previous article, msalminen at nphi.fi () says:

>Hi netters!
>
>I have had some trouble with my oligos, please help.
>
>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.
>
>Anyone with experience of such a check please respond. Help is very much
>appreciated.
>
>	Best regards, Mika Salminen
>
>		      msalminen at finnphi (Bitnet)
>		      msalminen at nphi.fi (Internet)
>
Mika-

Bad idea.

Reason 1:

The oligo is liable to soak out in the ethidium stain, leading to loss and 
cross contamination

Reason 2:  this gel is too little for good separation, though we use 16 cm
gels that work ok

The best way to localize the oligo band is with uv-shadowing.  The new
Maniatis (Sambrook) has a good description of this method, and how to
purify the oligo after soaking it out.

2 notes:  for uv shadowing they recommend a silica gel plate with uv
indicator... If you don't have one handy, many (but not all) intensifying
screens will work.  The best way to do this is have someone _show_ you;
I've seen folks struggle with no fluorescence.  When it works it's easy.

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

good luck,

dennis



More information about the Methods mailing list