Quick transformation protocol wanted

Jeremy Marcus marcusj at helix.mgh.harvard.edu
Thu Feb 13 14:01:23 EST 1997


The transformation protocol portion of the procedure outlined below is
well worth noting.  As far as I know, it can be performed with any
competent bugs (not just with cells prepared with CaCl2 a la Pope & Kent),
as long as the plates are warmed to 37 for 45 min to 1 hr prior to
plating.  While this admittedly doesn't save a _ton_ of time, the main
advantage is that, by eliminating the heat shock step, you gain an hour
that is free of obligations, so you can, for example, go to a talk or go
eat dinner while the plate is warming, and then come back and plate your
bugs.

Jeremy Marcus
Massachusetts General Hospital
Boston, MA

In article <5dvg7c$qfq at bioalp.biobase.dk>, wind at biobase.dk (Troels Wind) wrote:

: I haven't heard of what you describe, but alternatively you can
: try the protocol of Pope and Kent (Nucl Acid Res, vol. 24, 536-7, 1996).
: 
: In short:
: Harvest cells from 50 ml, OD=0.7-0.8.
: Chill on ice, resuspend in 25 ml ice-cold 0.1 M CaCl2.
: Chill on ice 30 minutes, harvest and resuspend in 2.5 ml 0.1 M CaCl2,
: include 10% glycerol if cells are to be stored at -80C.
: Store as 100 ul aliquots at 4C or -80C.
: 
: I dont remember for how long the cells are good at 4C, its in the
: original paper.
: 
: Transform by adding 1-10 ng DNA and incubate on ice for 5 minutes.
: 
: Plate on preheated plates.
           ^^^^^^^^^
*I've been told that best results come from warming the plates to 37 for 1 hr. 

: 
: I use this method when shuttling plasmids between my favorite
: strains, its not as quick-and-dirty as what you asked for, but it's
: the quickest I've heard of...
: 
: Troels Wind
: Aarhus University
: Denmark
: 
: A.P.N. de Boer (deboer at nefeli.imbb.forth.gr) wrote:
: : Hi,
: : Once upon a time, I learned a very quick (and dirty) transformation
: : protocol. It could be used to transform strains with plasmid DNA (which
: : doesn't need a high transformation efficiency). It was very quick and and
: : just entailed mixing some bacteria (colony from plate or pellet or
: : anything), DNA and the transformation mix and the mix could be plated.
: : I can't find the protocol anymore (moved labs). Does anyone here know of
: : such a protocol ?
: : Thanks, Thon de Boer



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