SSCP not single stranded.

D. KIM dkim at nmsu.edu
Fri Dec 6 00:01:36 EST 1996


In article <586d8s$2n0 at paperboy.ccc.nottingham.ac.uk> pdxrmb at vaxb.ccc.nottingham.ac.uk writes:
>Hi netters- here's a problem for all you SSCP buffs out there-
>
>I've been trying to do SSCP of restriction digested PCR products. The rest of
>the system is working well- the PCR is clean and the digests very neat (I'm
>labelling with P32 by spiking the amplification reaction) 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?- The DNA
>is very GC rich, but it amplifies superbly so I don't think denaturing it is
>the problem- it seems to be renaturing before I get it on the gel. I have
>heard that NaOH urea in the loading dye can prevent this, has any body any
>experience of this problem and its solution?
>
>Thanks in advance- Richard Badge.


Richard:

I can't remember exactly, but I tried a similar kind of experiment.  
After some effort, I stumbled on a review of PCR-SSCP in one of the first 
issues of _PCR Methods and Applications_.  In it the authors stated that 
SSCP is more sensitive in gels with very low %C.  After adjusting my gel 
formulation, everything went well.

What is the composition of your gel (and also other running conditions 
such as temperature of the run, etc)?

Daniel Kim
dkim at nmsu.edu



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