problem with sequencing in big dye v3

Roland Hübner roland.hubner at
Mon Oct 14 12:53:35 EST 2002

> > Now we don't do it any more, because with the TA cloning kit we have
> > about 10 colonies on 200-300 that are empty so...
> >
> >  Is that kit maybe based on topoisomerase? (5 mins "lig'n" can be too
> > much!)
> >
> > Roland
> Yes, this kit is effectively based on topoisomérase, and the ligation is
> very efficient.
> But what can be wrong with is for sequencing ??
> thank you for your answer
> eve

 Well, it is a TOPOisomerase... This catalyst can sometimes induce
a weird torsion (... with losses of [primer] sequence reported here 
earlier), so that for such templates the seq rxn is in trouble...

 In other words, I would clone the 'old' way to find out whether 
autoseqs work OK...

Bonne chance!

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