Chloroform colony lysis

Glen Shearer gshearer at whale.st.usm.edu
Thu Jan 5 15:25:40 EST 1995


Marc Goldstein (magoldstein at ucdavis.edu) wrote:
: In article <pemanuel-3012941104290001 at beaker.ab.umd.edu>,
: pemanuel at umabnet.ab.umd.edu (Peter) wrote:

: > Has anyone a good procedure for a cholorfom colony lysis?  The aim is to
: > lyse colonies expressing a recombinant protein and to transfer that
: > protein to a nitrocellulose filter for subsequent screening of the
: > proteins. Thanks
: >  Peter

: Peter-

:    A person working in my dissertation lab used to lyse E. coli (both K
: and B strains) by inverting the plate, and setting it on top of a beaker
: partially filled with chloroform.  I believe she used a thin-walled beaker
: of some sort that just barely fit the plate upside down.  There was a stir
: bar in the chloroform, and the whole arrangement was put on a stir plate
: for 5-50 minutes either at room temp or in the cold room.  She sealed the
: beaker/plate junction with parafilm to keep fumes inside to aid lysis and
 <<snip>>

How about placing the filter colonies up, on top of filter paper
soaked in NaOH then transfer to tris to neutralize?  Would this
not lyse the cells or would the colonies smear too much??

Glen
[gshearer at whale.st.usm.edu]




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