You can use an enzyme with an interrupted recognition site which leaves single-base overhangs (such as e.g. XcmI). If you design the intervening sequence properly, all you have to do is cut w/ XcmI and voilá! T overhangs a la carte.
Cheers, alejandro -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of [email protected] Sent: Wednesday, April 01, 2015 11:27 AM To: [email protected] Subject: Re: Home-Made TA cloning Am Sonntag, 9. Juli 1995 09:00:00 UTC+2 schrieb [email protected]: > I've been trying to think of a way to make my own TA cloning kit. > > There doesn't seem to be any available restriction enzymes that will > produce the proper ends for TA cloning (although I'm sure Invitrogen must > have one in their bag of tricks). > > I've been thinking that if I ran PCR on a linearized plasmid > (Bluescript), and used primers that annealed to each end of the plasmid > but had one or two loose A's hanging over the edge it might work. > > Any ideas out there. > > Cris Martin What about cutting with XhoI and filling up with Klenow but only using G T and C but no A for the filling up? Ben _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
