problem with sequencing in big dye v3
roland.hubner at ua.ac.be
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
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...
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