Thermal cycler sequecing...

Stuart Brown browns at ccu.umanitoba.ca
Wed Apr 8 16:06:15 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 tried cycle sequencing using the BRL kit which employs an end labelled
primer (kinase reaction, no cleanup steps) with moderate success.

I am now trying out the Vent Polymerase Kit from NEB with high hopes (IMHO these
guys don't sell something untill all the bugs have been worked out)

In both cases, I think a key factor is using a primer for sequencing that is 
internal to the primer used to generate the PCR fragment.  Also, the PCR 
product must be very clean (I cut bands out of a gel and then used Gene-Clean)

	Hope this helps  -Stu

-- 
Stuart M. Brown                               Postdocs rush in where wise men
U. of Manitoba, Dept. Plant Science, Canada       Fear to tread. 
browns at ccu.umanitoba.ca        
				Disclaimer?? Sure: "That Disc is Mine!"



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