plasmid recovery from "dead" plates

Rafa Maldonado Rafael at genetics.med.utah.edu
Tue Aug 23 18:11:53 EST 1994


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

>
> 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.  
> 

There is a similar protocol in Trend in Genetics, 10 (1994) 184. That I
used to do is: scrap as much of cells from the agar asyou can (small
bits of agar are OK, I think one colony won't be enough), resuspend in
TE buffer and ad same volume of phenol-chloroform. Precipitate with
EtOH and resuspend the pellet in several microliters of TE and make a
transformation with the total amount of DNA. Also, you can recover the
cells by spreading buffer in the plate, resuspending with a rod and
transfering the suspension to a tube.
Maybe PCR is a good idea, but I never tried.
Good luck!


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



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