Sequencing G-C rich DNA
Zhonglin.Chai at med.monash.edu.au
Tue Jul 14 21:39:51 EST 1998
Using ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with
AmpliTaq DNA polymerase, FS (Perkin Elmer), I successfully sequenced a GC
rich region (over 70% GC within 500bp containing a 100bp region with over
80% GC). The modified cycling conditions were 96 oC 15 sec, 58 oC 10 sec
and 72 oC 4 min, 25 cycles (PTC-100 Programmable Thermal Controller, MJ
Research Inc. USA).
ZhongLin Chai, PhD
Department of Pathology and Immunology
Monash University Medical School
Commercial Rd, Prahran, VIC 3181, AUSTRALIA
Telephone: (61 3) 9276 2698 (lab)
(61 3) 9276 2696 (office)
Fax: (61 3) 9529 6484
email: zhonglin.chai at med.monash.edu.au
>Hayley - The Comet wrote:
>> Does anyone have a method for sequencing G-C rich cloned DNA?
>> The sequencing PCR conditions we are currently using for all > sequen=
>cing reactions regardless of nucleotide sequence is :
>> for 25 cycles.
>> Please let me know....
>I have never done sequencing PCR, but I wonder if 96'C for only 10 sec
>would be sufficient for G-C rich sequences to dissociate.
>Would a high GC-content dictate a longer time at 96'C as in a regular
>Patrick F.H. Lai < PFHLai at looksmart.com >
>Grad Student, U of Toronto
>LookSmart =85 or keep looking.
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