Question on SSCP

Hui Yuk Fun wayhui at hkusub
Tue Apr 25 07:10:51 EST 1995


I'm new in performing SSCP. In the latest trial I loaded the undenatured
PCR product samples (<300 bp) beside the denatured samples to see if the
samples're fully denatured. In the X-ray film I found three bands in the
undenatured samples and two bands in the denatured samples. Two of the
bands from the undenatured samples corresponded to that in the denatured
samples, the extra band either ran faster or slower than the other two
bands. I think the extra band is the undenatured PCR product. The
questions are: 

i. Do the undenatured PCR product migrates faster or slower than the
   ssDNA with a stable secondary structure? Is there any definite
   answer?

ii. Why is there 3 bands in the denatured samples. Is it possible that
    the loading dye denatured the PCR products? The loading dye is used
    contain 95% formamide and 0.1% SDS and dye. I diluted the 10ul PCR
    mixture 10 times with the loading dye.

Thanks for any help.
Wayland


--------------------------
Wayland Yuk-Fun HUI, MPhil 
Department of Paediatrics,
Faculty of Medicine, 
The University of Hong Kong.

Pager    : 1128631 A/C 7260
E_mail   : 1) wayhui at hkusua.hku.hk
           2) wayhui at hkucc.hku.hk
Telephone: (852)-2855-4898
Fax      : (852)-2855-1523





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