Cycle Sequencing

Jun Ishikawa jishikaw at
Thu Jun 3 13:48:20 EST 1993

Dear netter,

I am trying to sequence Streptomyces DNA (GC%=>70%) with USB cycle
sequencing kit. But I can not obtain only very weak signals. 
My protocol is as follows:

   Labeling reaction: 95c, 15sec
                      60c, 30sec
                      x 30 cycles
Termination reaction: 95c, 30sec
                      72c, 120sec
                      x 30 cycles.

Double-stranded DNA (upto 2-kb) inserted in pUC plasmid were subjected
to sequencing.

Does anyone have any idea or suggestion about the cycle sequencing of 
high GC content DNA?

Thank you in advance.

   Jun Ishikawa, Ph.D. (Mr.)     Dept. of Bioactive Molecules
                                 NATIONAL INSTITUTE of HEALTH
   jishikaw at       1-23-1, Toyama, Shinjuku,
                                 Tokyo 162, Japan
P.S. Sorry for my poor English.

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