Cycle Sequencing

Jun Ishikawa jishikaw at ddbj.nig.ac.jp
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 ddbj.nig.ac.jp       1-23-1, Toyama, Shinjuku,
                                 Tokyo 162, Japan
 ==============================================================
P.S. Sorry for my poor English.



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