Need advice on dT tailing of blunt end cut pBluescript

Bernard Murray bernard at
Wed Apr 16 17:26:38 EST 1997

In article < at>, at says...
>Dear All,
>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 (National Cancer Institute, NIH, Bethesda MD, USA)

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