Construction of TA cloning vector

Dr. C. Dolphin cdolphin at crc.ac.uk
Tue Feb 1 09:42:16 EST 1994


In article 9699 Helge Weissig <helgew at ljcrf.edu> writes


>>you could also incubate blunt-cut vector with dideoxyTTP (ddTTP) and terminal
>>transferase which will add single ddTTP to each 3'end. Addition of extra dATP
>>in the final elongation stage of PCR may also increase proportion of products
>>with A overhangs (question - does Taq add just a single dATP or does it also
>>have terminal transferase-like activity?). We have used this for succssefully
>>for sub-cloning of many PCR products. Also include a touch of the enzyme used
>>to blunt cut vector in the ligation reaction to reduce background.
>>
>>Colin

>REALLY?????

>how come that you get ligation to a ddTTP tailed DNA strand?

>how come that you get ligation to a ddTTP tailed DNA strand?

>puzzled,
>helge

Dear Helge, obviously you don't get ligation on that strand but you will on the other one at that position, ie. you get a sort of double nicked (half-ligated)
plasmid. This seems to be fine for transfection and then the bugs produce super
-coiled. We've used Taq to do the same thing but with dTTP rather than ddTTP
and this also works but how efficient is this activity of Taq ? 

Colin

Colin Dolphin				Email: c.t.dolphin at qmw.ac.uk
Biochemistry				Tel: (44) 71 975 5555
Queen Mary & Westfield College	FAX: (44) 81 983 0531
London E1 4NS, UK



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