TA cloning vector

Stuart Brown browns at ccu.umanitoba.ca
Fri Aug 6 14:28:57 EST 1993


In article <1993Aug5.154952.26316 at news.unige.ch> mike at medsun.unige.ch (Mike Morris) writes:
>  We have recently worked through a lot of approaches to PCR cloning 
>(we used to be able to do this, but then the weather changed, so no
>more clones).
>  Making T vector yourself is possible, but seems to invovle some luck -
>a while ago, the consensus was: try it in big batches, so if it works
>you have plenty!
>  We have just tried the "second generation" Invitrgoen kit. I got the
>same result as with the first generation - nothing doing.
>  A Stratagene/Bluescript kit is in the offing, but is not yet available in
>Europe.
>****************************
>Mike Morris PhD      
>Division of Medical Genetics       
>Geneva                
>Switzerland
>****************************  

I have had mixed success with the Invitrogen kit.  The secret seems to 
be choosing the right colonies off of your kanamycin plate.  Not the
white ones, not the blue ones, but the sort of whitish ones with tiny
blue dots in the center.  I don't know why this is, but there is some
mumbling in the kit's instructions about out of frame religation of the
vector forming white "background" colonies.  I also get widely different
numbers of colonies from different ligations done with different PCR bands
cut from the same gel.  Nevertheless, this is the best method that we have
found for cloning PCR products, since we cannot add restriction sites to 
all of our primers.  

I can now relaible go from PCR to colonies in one day, mini-preps and
sequencing on the next day!

	-Stu

-- 
Stuart M. Brown                             If you can remain cool when all 
U. of Manitoba, Dept. Plant Science         Around you are in panic,
Winnipeg, Manitoba, CANADA
browns at ccu.umanitoba.ca            Then you surely misunderstand the situation



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