buzardg at MAIL.NCIFCRF.GOV
Wed Jan 21 17:10:05 EST 1998
I am just starting out doing SSCPs visualized through
silver staining. Although I get good, clear bands on the
gel I do not seem to have 2 bands indicative of the
strands (I am working with mtDNA). The single bands seem
to show the type of variation I am after but I am
worried about there not being two bands. Is this most
likely due to:
1) Incomplete denaturation (I use 3 min at 94oC after
adding 1 vol of 95% formamide).
2) Renaturation. I keep on ice prior to loading (perhaps
15 min) but gel is run at room temp).
3) Migration of single strands off gel, and I am seeing
something else. However I see very faint bands near the
top of the gel which I assume are the double stranded
DNA so I can't see what else I could have.
Any ideas anyone? Thanks Craig. (BGYCSW at LEEDS.AC.UK)
It could be renatured DNA, depends on the sequence and the concentration of
DNA, and the efficiency of your denaturant. The standard ploy to test this
is to run an undenatured dilute molecular weight marker in one lane, an
undenatured sample in another, and the denatured SSCP samples in the
remaining lanes. As a rule of thumb, the single bands run above the dsDNA
at twice the apparent size of the dsDNA.
Other reasons that you can have a single SSCP band:
Co-migration of both. Unlikely in your case, since you are seeing some
shifts, and they would not both move in the same way.
Asymmetric PCR, due to bad or insufficient primer, or mismatched Ta of
Fuzz out, a phenomenon where at a given run condition one strand does not
have a defined conformation and thus does not run as a tight band. See
papers on transverse temperature gradients for SSCP analysis where this
effect is nicely illustrated.
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