rescue dead stock

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Thu Sep 1 12:42:21 EST 1994


 In article <atesg.2.2E639C28 at asuvm.inre.asu.edu>
 atesg at asuvm.inre.asu.edu (elliott s. goldstein) writes:

| does anyone have a proceedure to extract plasmid DNA from a dead E. coli 
| stock? It is frozen in 40% glycerol. Thanks.

Sure. Spin the cells out in an eppendorf tube and follow the coli-blaster
protocol...The use of 1M NaCl to prepare the cells for osmotic shock
increases the plasmid DNA recovered and you can transform by directly
adding comptetent cells to the tube. It worked for me on several occasions
to recover a lost clone and I hope it works for you too.

    *** Paul's "coli-blaster" mini-prep method for Tons O' DNA *** 
 
1.  Pellet the cells in a clinical centrifuge 5 min. or eppendorf for
    2 min.
2.  Pipet off the sup. and resuspend the pellet in 1.0 ml of 1 M NaCl 
    (Note: This is optional, but I find it helps if the bacteria
    produce excess polysacharides or if the cells are quite old. This
    should be done if plasmid DNA is to be extracted from non-viable
    cells removed from agarose plates or glycerol stocks.) Leave the
    suspended cells on ice from 10-60 minutes, depending on whether it's
    a coffee break or lunchtime ;-)
3.  Transfer the mixture to a 1.5 ml centrifuge tube (if you haven't yet).
4.  Spin 1 min. in the table top centrifuge, decant sup.
5.  Resuspend the pellet in 100 ul of 25 mM Tris, 10 mM EDTA, pH8.0
6.  Squirt in 200 ul of pre-warmed (65 C) 0.2 M NaOH, 1.0% SDS and
    flick the tube two or three times by snapping with your fingernail.
7.  Place the tubes on ice for 10 min. Most of the time lysis occurs
    immediately and you can move on to the next step after doing it to
    all the tubes.
8.  Mix in 150 ul of 3 M Na acetate pH 4.8 by flicking the tube 
9.  Place on ice for 15-20 min., spin 15 min. at 4 C in an eppendorf
    centrifuge.
10. Remove the sup. and transfer to a clean tube.
11. Mix in 125 ul of 10 M Ammonium acetate 
12. Add 1.0 ml of 100% ETOH, invert the tube, and place at -20 C [can be o/n]
13. Spin 15 min. at 4 C  (For recovery of DNA from non-viable cells, dry
    the pellet in a rotovac and add 200 ul of ice cold competent cells for
    transformation directly -> do your normal transformation protocol by
    heat shocking for 2 min. at 42 C, expressing at least 2 hours at 37 C,
    and plating the entire amount of cells onto selective media.)
14. For restriction analysis, remove the supernatent and wash the white
    pellet by resuspending it in 100-200 ul 70% ETOH. Spin again.
15. Repeat #15 two more times
16. Dry the pellet in a rotovac and resuspend the DNA in 20 ul of sterile dH2O
17. Use 3-5 ul per 20 ul restriction digest reaction in 20 ul total volume.
 
References:

@article{Birnboim1979,
author = "H. C. Birnboim
     and J. Doly",
title = "A rapid alkaline extraction procedure for screening
recombinant plasmid {DNA}",
journal = "Nucl. Acids Res.",
volume = "7",
pages = "1513-1523",
year = "1979"}

@article{Schwinghamer1980,
author = "E. A. Schwinghamer",
title = "A method for improved lysis of some
Gram-negative bacteria",
journal = "FEMS Microbiology Letters",
volume = "7",
pages = "157-162",
comment = "osmotic shock for lysis",
year = "1980"}

@inbook{Kuykendall1988,
author = "L. D. Kuykendall
     and P. N. Hengen",
editor = "N. S. Subba Rao",
title = "Biological Nitrogen Fixation - Recent Developments",
chapter = "Microbial genetics of legume root nodulation
and nitrogen fixation",
publisher = "Oxford and IBH Publishing Co. Pvt. Ltd.",
address = "New Delhi",
pages = "71-111",
comment = "osmotic shock for lysis using 1M NaCl; pages 77-79",
year = "1988"}

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
* /Hey, Cool! -> http://fconvx.ncifcrf.gov:2001/~pnh/info.html <- Hey, Cool! /*
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