which restriction enzyme should I use to digest the vector? - (Oct/19/2006 )

I've been constructin an cloning vector recently (not for commercial use), and this vector will be used for cloning PCR product. This vector is something lis Invitrogen's Topo TA Kit.

The question is, how can I generate an stick end with a "T" project, and the sequence before this T
is "CCTT",which can be recoganized by vaccinia topoisomerase?


Thanks a lot.

I've been constructin an cloning vector recently (not for commercial use), and this vector will be used for cloning PCR product. This vector is something lis Invitrogen's Topo TA Kit.

The question is, how can I generate an stick end with a "T" project, and the sequence before this T
is "CCTT",which can be recoganized by vaccinia topoisomerase?


Thanks a lot.

QUOTE (Ocras_ren @ Oct 19 2006, 05:12 AM)

I've been constructin an cloning vector recently (not for commercial use), and this vector will be used for cloning PCR product. This vector is something lis Invitrogen's Topo TA Kit.

The question is, how can I generate an stick end with a "T" project, and the sequence before this T
is "CCTT",which can be recoganized by vaccinia topoisomerase?


Thanks a lot.

do u need a 'T' sticky end on both sides ?

try an enzyme like AhdI (NEB). This could help u to have ends with a single 'T' only.