SSCP not single stranded.
jmacfarl at hgmp.mrc.ac.uk
Thu Dec 5 09:37:02 EST 1996
On 5 Dec 1996 pdxrmb at vaxb.ccc.nottingham.ac.uk wrote:
> But when I try to
> do SSCP (i.e. add 95% formamide loading dye, boil (10 minutes) , chill on ice
> and load on a non-denaturing gel, I get pretty much the same pattern as if I'd
> not boiled the sample- i.e. most of the sample is in the form of DNA that
> migrates with double stranded digest bands. What am I doing wrong?
> I have
> heard that NaOH urea in the loading dye can prevent this, has any body any
> experience of this problem and its solution?
I have tried NaOH denaturation. With radioactive samples it worked well.
The original reference that I followed (from Technical Tips in Trends in
Genetics Vol 8 no 2 page 49) used a final concentration of 0.05 M NaOH and
1mM EDTA. However I was advised (I forget by whom) that this was too low
and used a final NaOH concentration of 0.2M. Interestingly when I tried
NaOH denaturation on non-isotopic silver-stained gel the single-strand
resolution was terrible.
I agree that denaturation is unlikely to be a problem. We denature for
less than half the time that you do (4 mins) and have no problems. I chill
the buffer in the top resevoir to 4 degrees C. This does not seem to make
a huge difference and I doubt that it would solve your problem.
Do you clean the digested DNA before running it on the gel? We have found
that this makes a huge difference to resolution.
I have had fragments that have produced useless (smudgy, very weak
bands) single strands at room temperature but have resolved fine when run
at 4 degrees C.
Just a few thoughts....
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