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Home-Made TA cloning

Alejandro Miguel Martin Dunn via methods%40net.bio.net (by alejandro.martin from cigb.edu.cu)
Wed Apr 1 16:23:24 EST 2015


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: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of anatomie.zebrafisch from gmail.com
Sent: Wednesday, April 01, 2015 11:27 AM
To: methods from magpie.bio.indiana.edu
Subject: Re: Home-Made TA cloning

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?

Ben
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