Chloroform colony lysis

Sebastian W. Bunka seb at i102pc1.vu-wien.ac.at
Tue Jan 3 06:43:53 EST 1995


Peter (pemanuel at umabnet.ab.umd.edu) 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
I think this is described in several mol.bio. books like Sambrook/Maniatis
and so on..... (See colony lift/plaque lift).

I use the following procedure for screening colonies for expression of
recombinant proteins:
- transform your E. coli comp. cells with your ligation mix, grow overnight
- prewarm LB-Ampicillin plates for 2 h at 37 degC (Lid lightly opened)
- spread 50 mikroliters IPTG (100 mM) over the warmed plates
- get the plates with the transformed E. coli
- with 2 forceps place a dry NC membrane on to the surface with the colonies
- Wait until the NC membrane is completely wet, and mark the position
  of the membrane with needle and ink
- lift mebrane off the plate and put on the LB-Ampi + IPTG with colonies UP
- inkubate 2h at 37degC
- prepare glass tray (about 20cm x 20cm x 5-10 cm high), put several
  pasteur (glass) pipettes on the bottom and fill in some
  chloroform, so that the NC mebranes don't come in direct contact
  with the chloroform !; cover with saran wrap and wait about 15 minutes.
- lift the NC from the IPTG plate and place it on the pasteur pipettes
  (colonies UP).
- incubate in the chloroform vapor for 15 minutes
- remove the NC from the tray and let evaporize the chloroform from
  the membrane (never place it fresh in a plastic tray !!)
- place the membrane in a petri dish and cover w/ blocking solution (I use
  0.5% gelatine in tris buffer w/ 0.05% Tween 20).
- incubate 2 hours on a shaker
- SCRUB OFF remaining colony material with gloves while the NC immersed
  in the blocking buffer !!!!!
  Otherwise you'll get false positive results.
- proceed with detection ....

Hope this helps,
Sebastian
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