Thermal cycler sequecing...

John Nash num208jn at MBDS.NRC.CA
Wed Apr 1 12:01:03 EST 1992


In article <9203312113.AA25586 at umailsrv0.UMD.EDU>, William_D_WARREN at UMAIL.UMD.EDU (ww40) writes:
>Does anyone out there get good results from thermal cycler sequencing ?
>
>We have been trying to sequence gel isolated PCR products using
>a gel purified end labelled primer (one of the PCR primers) and
>can't get clean sequence. We've tried 3 step and two step cycling
>with various time regimes and temperatures and still no luck.
>
>Has anyone had better luck using this technique ???

I have never used Taq polymerase based cycle sequencing, but I have
had excellent results sequencing a 1 kb PCR product with New England
Biolab's CircumVent sequencing which utilises exo-minus Vent
polymerase.  I use their standard reaction protocol but cycle at
95/1min; 55/1min; 72/1min; twenty times after a hot start.

My product was excised from an agarose gel, cleaned up with Glass
Milk, and eluted into water. 

If you want further details... email me.

P.S.  Usual disclaimer...


John Nash     (Internet:) Nash at biologysx.lan.nrc.ca.  
Email to my other addresses is forwarded automatically,
Disclaimer:  All opinions are mine, not NRC's!



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