Cycle sequencing - no product

Lisa Bibbs lisa at scripps.edu
Fri Sep 9 14:14:28 EST 1994


In article <34ms1l$eb0 at arco2.cd.afrc.ac.uk>, partis at arcu.afrc.ac.uk
(partis) wrote:

> I have a colleague who has been cycle sequencing.  He is having a problem
> with his current attempts using the ABI DyeDeoxy Terminator kit.  The PCR 
> product was cloned into pCR-Script SK(+) vector (Stratagene)and cycle
> sequenced using T3 or T7 primers (Genosys).  The problem is that he finds
> no reaction product. Has anyone had similar experiences.  Possibilities
> which he has raised are: non-optimal template amount or  inappropriate 
> primers.  Any help from fellow Netters would be much appreciated.  Comments 
> by posting or e-mail to me, please - I will forward.
> 
> Mick
> -- 
> 
> M.D.Partis, Molecular Biology, HRI
> Tel:  0903-716123                  Internet e-mail: PARTIS at afrc.ac.uk
> FAX:  0903-726780                  X.400: S=partis,O=afrc,P=uk.ac,C=gb

How was the sample prepared for sequencing? Try the Quiagen preps.  Did the
control cycle sequencing reaction work?  You can use those primers on the
pgem  that comes with the kit.  That would answer your primer question.  I
don't know the pCR-Script but if it is anything like KS- then it should
have those priming sites although we prefer to use the M13 forward and
reverse.  Also, do you have TE in your sample or the primers?   In the
cycle sequencing with the Dye terminators, TAQ requires a certain
concentration of magnesium and if you have TE in there that will throw off
the delicate balance. TAQ is fussier in with the Dye labelled terminators
than it is with normal PCR reactions.

Good Luck
Lisa Bibbs 



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