Actually, cloning in the PCR fragments works beautifully for TA cloning exactly for the reason you stated - overhanging As. I have used that method before. The only issue is, you would have to test for the correct orientation of the fragment!
Hope that helps, Pow On 1 April 2015 at 11:26, <[email protected]> wrote: > 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
