Home-Made TA cloning

[Srinivas K. Janardan, M.D.]

In article <3ttsks$6mf at gazette.bcm.tmc.edu>, Brian Desany
<bd033054 at condor.mbcr.bcm.tmc.edu> wrote:

 In our lab, I developed a plasmid from pBluescript that has an additional
30+ bases in it at the MCS that contains two XCM I sites.  The additional
insert is designed to yeild two T single base overhangs after XCM I
digest.  This is then ready to use for TA cloning.  I tried it a few times
but it did not appear to work that well.  Right now we are using the
invitrogen kit.  Someday I might pull this plasmid out of the freezer and
trry it again. BTW I have no connection to invitrogen.

> There are restriction enzymes that leave single base overhangs in the 
> middle of their site, XcmI (ccannnnn/nnnntgg) for example.  Two of these 
> sites with the appropriate bases chosen for the "n"'s should make it 
> really easy to TA clone - you don't have to ligate anything to the 
> cleaved vector before you do your actual TA cloning reaction.  Although 
> I haven't got around to trying it yet.