Losing pieces of plasmid

Bernard Murray murrayb at dc37a.nci.nih.gov
Fri Aug 11 09:07:53 EST 1995


In article <199508101942.OAA24812 at borcim.wustl.edu>, brett at BORCIM.WUSTL.EDU 
says...
>
>>Does anyone have a suggestion that will keep my E. coli from spitting out 
>>pieces of plasmid?  I'm trying to put 6kb into a 3kb vector that already 
>>has 10kb inserted.  Clones isolated after the ligation don't look like 
>>either the parent or the predicted product.  I know that 19kb is pushing 
>>it as far as plasmid size, but I'd appreciate any advice.
>>Thanks
>>MLK.

>It can be done. Use a recA host, of course. Design your ligations to be
>very efficient (who doesn't?! ;-)) - no, but don't count on a double blunt
>end ligation to jump from 13kb to 19kb. I have had success in 17kb
>constructions, and I'm sure you can too. Godd luck!
>Brett Lindenbach
>brett at borcim.wustl.edu                             

I've also heard good reports about Stratagene's SURE strain.  Although I've
used it myself I have never tried the same constructs in other strains so
I don't know how much advantage this confers.
	Also, is there any chance that your plasmid is expressing something
toxic - this would increase the chance of recombination?  Maybe refraining
from over-growing cultures would reduce the events.
		Good luck
			Bernard
Bernard Murray, Ph.D
bernard at elsie.nci.nih.gov  (National Cancer Institute, NIH, Bethesda MD, USA)




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