SSCP not single stranded.

pdxrmb at vaxb.ccc.nottingham.ac.uk pdxrmb at vaxb.ccc.nottingham.ac.uk
Thu Dec 5 06:56:12 EST 1996


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.



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