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,
On 1 April 2015 at 11:26, <anatomie.zebrafisch from gmail.com> wrote:
> Am Sonntag, 9. Juli 1995 09:00:00 UTC+2 schrieb s06... from aix1.uottawa.ca:
> > 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?
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