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:
>I have had some trouble with my oligos, please help.
>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
> Best regards, Mika Salminen
> msalminen at finnphi (Bitnet)
> msalminen at nphi.fi (Internet)
The oligo is liable to soak out in the ethidium stain, leading to loss and
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?
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