Creating T-cloning vector

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Thu Feb 18 04:36:52 EST 1999


In article <36CC4D6D.761A at cardiff.ac.uk>, Dr Antony Halsall
<halsalla at cf.ac.uk> writes
>Hi
>
>I am trying to produce a T-cloning vector for the direct ligation of a 
>PCR product into a reporter plasmid (pGL3-basic). I know it is possible 
>to create the necessary 3' T overhangs by incorporating two adjacent 
>restriction sites for either HphI, AspEI or XcmI, however all these 
>enzymes will cut pGL3-basic. So my question is does anyone know of 
>another enzyme that will allow the creation of 2 3' T overhangs or a 
>method for cutting at a site present in the pGL3-basic and filling in to 
>create the appropriate overhang?
>
>Thanks in advance
>
>Dr A. Halsall
>Dept. Psychological Medicine
>UWCM

Blunt your vector. Either incubate with Taq pol and just dTTP or use
terminal transferase and ddTTP. All in appropriate buffers for the
enzyme in question

Duncan

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Duncan Clark
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