plasmid recovery from "dead" plates

Eric C. Anderson anderson at pharmdec.wustl.edu
Wed Aug 24 09:45:36 EST 1994


In article <33d3bq$5d5 at emoryu1.cc.emory.edu>, kwinkel at cc.emory.edu
(Kimberly Ann Winkeler) wrote:

> I am trying to recover some plasmids from what are apparently dead 
> colonies--colonies picked and inoculated into liquid media do not grow.  
> I have tried the Shuldiner and Tanner method for recovery of plasmids 
> from nonviable colonies (Biotechniques 12(1) p66 (1992)) which involves 
> picking a colony, boiling it in water, and transforming some of the 
> supernatant.  I tried this method with four different clones with no 
> success.  
> 
> Has anyone tried this method (or any other method) for recovering 
> plasmids with any success?  Does anyone have any tips or suggestions?  
> I'd really like to recover these plasmids if possible since trying to 
> reconstruct them will be a major pain in the butt!!!
> 
i don't know if the protocol which i use has ever been published.  since
it's so low tech i doubt it, but i'm sure that somebody else must have
tried it.  i've gotten both good and bad results with this but most of the
time i am able to recover my transformants.

i just take the plate with the "dead" bugs on it, and use the wide end of
an autoclaved pasteur pipet to take a plug out of the most dense portion of
the bugs on the plate.  i then grow this plug in 100ml of 2xYT or Terrific
Broth (both have worked in the past, i tend to use whichever one is on my
bench) onvernight at 37 with shaking (like any normal culture).  be sure to
include whatever antibiotic you normally use in slightly greater
concnentration (1.5X of normal is what i use) and let them grow to an OD260
of at least .8-1.0.

then use these bugs to streak out new plates (do 2 or 3 plates just to make
sure) and these bugs should be your transformants.  i've also tried doing
minipreps from these cultures directly with mixed results and i don't
really reccomend it.  once you plate out the bugs and get colonies you just
proceed normally as if nothing ever happened.

good luck,
eric
-- 
*************************************************
*there's too much blood in my caffeine system!  *
*************************************************
eric c. anderson
dept. of molecular bio. and pharm.
washington univ. school of medicine
660 s. euclid box 8103
st. louis, mo 63110

(314)362-3963 (lab)
(314)862-2435 (home)
(314)362-7058 (FAX)



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