optimizing CIP

Rafa Maldonado Rafael at genetics.med.utah.edu
Mon Jan 31 18:54:44 EST 1994


Some suggestions more:

1- Dissolve your DNA in water, not in TE. Sometimes, Zn is chelated by 
buffers with too much EDTA. Zn is necessary for the activity of the
enzyme. Indeed, the  reaction buffer has no EDTA. I think that this
buffer with triethanolamine is more delicate than CIP itself. Check the
age of both; older than 6 months is bad.
2- Some CIP can be stored at -20, other not. Have you checked that?
Boehringer's one cannot be frozen; Promega's can. How do you store your
CIP?
3- As control of your plasmid, you can use bacterial phosphatase
alkaline, but you can't use the resultant vector for cloning (some
years ago, I used this enzyme, and it's really difficult clone
anything). It works much better than CIP, but It's impossible to take
it away from your DNA. Anyway, it can be useful to know if your vector
is OK (I mean, no many undigested molecules).
4- It's a good idea follow the Maniatis' protocol (as always!)
5- Do you get transformants without ligase in you cloning reactions?

Good luck and don't give because a single thing doesn't work!

----------------------------------
Rafael Maldonado
Howard Hughes Medical Insitute
University of Utah
Rafael at genetics.med.utah.edu



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