Sequence problems

Karen Usdin ku at helix.nih.gov
Tue Mar 23 11:12:08 EST 1999

It sounds as if you have a major secondary structure problem so that few
of your products are making it past the central region (this would be
visible as bands in all four lanes if you were using endlabeled primers
or deoxynucleotides and left out the TdT). Cycle sequencing might help
as would reducing the cation concentration in your buffers, minimizing
any time for reannealing primers (in Sequenase reactions we just
coprecipitate the denatured template and primer and then resuspend and
sequence immediately, a annealing step at 37 just makes things worse).
You might also get better results if you PCR'd your insert up using
7-deazadGTP instead of dGTP if your secondary structure involves
non-Watson-Crick e.g. G-G base pairs or dITP if it involves G-C base
pairs and then sequenced the PCR product.

Karen Usdin
Senior Staff Fellow                  
Section on Genomic Structure and Function
Laboratory of Molecular and Cellular Biology, 
National Institutes of Diabetes and Kidney Diseases
Building 8, Room 202                       
8 Center Dr MSC 0830
National Institutes of Health
Bethesda, MD 20892-0830

Tel: 301-496-2189
Fax: 301-402-0053
email: ku at helix.nih.gov

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