Sequencing G-C rich DNA

Zhonglin Chai 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
Alfred Hospital
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:
>
>> Hi
>> 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 :
>=0996'C=0910 sec
>=0950'C=095 secs
>=0960'C=094 mins
>> for 25 cycles.
>> Please let me know....
>..................................................................
>
>Dear Hayley,
>
>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
>PCR ?
>
>Just curious.
>
>
>Patrick F.H. Lai  < PFHLai at looksmart.com >
>Grad Student, U of Toronto
>
>LookSmart =85 or keep looking.
>http://www.looksmart.com





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