TA cloning ratio

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Sun Mar 19 13:12:44 EST 1995


In article <3kfg5l$gv8 at mark.ucdavis.edu>, ez022056 at dale.ucdavis.edu (Edward Wang) writes:
>I am currently using the Promega p-GemT system and having lots of 
>problems with it.  I am not getting colony growth.  My guess is that the 
>insert to vector ratio is too low (They recommend 1:1 ratio).  
>Has anyone who used this kit any 
>suggestions?  Right now, my controls are not working either (a insert 
>which they provide).  Also, is it myth or fact that for cloning, one 
>should use fresh PCR product because the overhangs fall off?  Any 
>suggestions would be appreciated.
>
I am a happy user of this kit for more than 1.5 years, and it always worked
for me. You did not mention which competent cells you are using. In the cloning
experiment of this kind one needs fairly good cells, and the company
specifically says this in the accompanying booklet. Try transforming your cells
with 1 ng of any uncut vector. If you get less then 1000 colonies, they are not
good for T-vector cloning. With proper cells, the number of colonies from the
vector, ligated in the absence of an insert is never zero.
 

*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
Universitaetstr.16
CH-8092 Zurich
"Some ideas are obscure not because they are complex, but because they 
 are excluded from our circle of comprehension" - Kozma Prutkov



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