Need advice on dT tailing of blunt end cut pBluescript
bernard at elsie.nci.nih.gov
Wed Apr 16 17:26:38 EST 1997
In article <iain.shaw-1604970938170001 at pc0776.irad.bbsrc.ac.uk>,
iain.shaw at bbsrc.ac.uk says...
>I am currently cloning PCR reagents into invitrogen TA kit, quite
>succesfully but at quite a high cost. What I would like to do is make my
>own by cutting pBluescript and add a single T overhang. I have a
>T-tailing kit from Boehringer (DNA Tailing Kit), but you do not appear to
>be able to limit the amount of Ts that are added. Does anyone know a way
>of adding a single T this way, or has anyone any suggestions on how else
>to do this.
If I remember correctly this is in the faq (Paul?).
I don't know what the Boehringer kit contains (probably TdT as the
enzyme) so I can't comment on adapting that kit but (again, if I
remember correctly) you can either tail the blunt cut (EcoRV, HincII,
SmaI etc.) or cut and polished vector with Taq and dTTP (as this will
only add a single T) or instead use an end labelling enzyme and ddTTP
(as this cannot extend after adding the first T).
Sorry, I don't have the faq to hand but this should at least
get you started.
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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