TA cloning kit problems

Timothy Bushnell bushnt at rpi.edu
Wed May 11 08:57:39 EST 1994


In Article <2qov7iINN4i2 at newsstand.cit.cornell.edu>, douglas l feinstein
<dlfeins at cumc.cornell.edu> wrote:
>Recently we have had serious problems using the Promega TA cloning kit.
>Up to recently, we were very happy with it, good amount of recombinants,
>low background. The last few batches of vector we received contain a
>contaminant that gives white colonies on  Xgal plates, but does not have
>any inserts. We sequenced a few and identified the contaminant as a
>pT7SP6319, and older vector from Pharmacia. 
>
>        Until this problem is solved, I'd like to find out other sources for a
>good TA cloning kit. Also, there was a TA vector developed by someone and
>being made available, posted here about a month ago. I assume that it was
> pac1 cuttable vector that would yield T overhangs, or some related
>enzyme. If this is still available, I'd like to get an aliquot.
>

    In our lab we have used both the TA cloning kit from Invitrogen and the
TA kit from Novagen.  In both cases, we have gotten excellent results.

    The Invitrogen kit is excellent, but is a bit expensive for routine use.  

    The Novagen system is nice.  You can buy cut prepared plasmid alone and
then use your favorite cells instead of the "NovaBlues" that they provide. 
In fact, I would not recommend using their prepared competent cells at all. 
We usually get a large number of satellite colonies, unless we up the
ampicillin concentation to 100 ug/ml.  However, if you streak out their
cells and make them competent by any of a number of protocols, we have found
that we get excellent transformation frequencies, and do not have the
satellite colony problem any more.

    I hope that this helps you.

Timothy Bushnell
Rensselaer Polytechnic Institute
Plant Research Group

    These in days when heavens mouring/ the days when earths foundation shook/
followed their mercenary calling and took their wages and are dead.
    Their shoulder held the sky suspended/ they stood and earths foundation
stayed/  What god abanded, these defended/  And saved the sum of things for pay



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