Inoue 1990 original paper for SEM transformation

Jayakumar, R via methods%40net.bio.net (by R.Jayakumar from roswellpark.org)
Wed May 2 08:08:41 EST 2012


You can also read the Current protocols in Molecular Biology. It is in there.
Jay


-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of DK
Sent: Tuesday, May 01, 2012 4:36 PM
To: methods from magpie.bio.indiana.edu
Subject: Re: Inoue 1990 original paper for SEM transformation

In article <7808145.140.1334065926763.JavaMail.geo-discussion-forums from vbla14>, Bjorn <bjornjobb from gmail.com> wrote:
>Hi all,
>does anyone have the original paper for SEM transformation:
>
>Inoue H, Nojima H, Okayama H. 1990. High efficiency transformation of 
>Esche= richia coli with plasmids. Gene, 96: 23=9628.
>
>http://www.sciencedirect.com/science/article/pii/037811199090336P
>

>From that paper, copy/paste: 

(a) Standard protocol for the preparation of competent cells by SEM Media (SOB, SOC and LB) were prepared as described by Hanahan (1983). For selection of transformed E. coli, LB plates containing 50/~g Ap/ml were used. To make TB
(10 mM Pipes/55 mM MnCI2/15 mM CaC12/250mM KCI), all the components except for MnCI 2 were mixed and the pH was adjusted to 6.7 with KOH. Then, MnCI 2 was dissolved, the solution was sterilized by filtration through a preripsed 0.45 pm filter unit and stored at 4°C. All salts were added as solids.
Frozen stock (in LB/7~o DMSO) DH5 cells were thawed, streaked on an LB agar plate, and cultured overnight at 37°C. About ten to twelve large (2-3 mm in
diameter) colonies were isolated with a plastic loop, inoculated to 250 ml of SOB medium in a 2-liter flask, and grown to an A6o o of 0.6 at 18°C, with vigorous shaking
(200-250 rpm). The flask was removed from the incubator and placed on ice for 10 min. The culture was transferred to a 500ml centrifuge bottle and spun at 2500 xg . 3~00 rpm in Beckmann J-6B centrifuge) for 10 min at 4°C.
The pellet was resuspended in 80ml of ice-cold TB, incubated in an ice bath for 10 min, and spun down as above. The cell pellet was gently resuspended in 20 ml of TB, and DMSO was added with gentle swirling to a final concentration of 7~o. After incubating in an ice bath for
10 min, the cell suspension was dispensed by 1-2 ml into tissue-culture cell-freezing tubes and immediately chilled by immersion in liquid nitrogen. The frozen competent cells were stored in liquid nitrogen for at least one month without a detectable loss of competence.

(b) Standard protocol for SEM transformation A tube of competent cells was thawed at room temperature, dispensed by 200/~1 into 15 ml polypropylene tubes (Greiner, F.R.G.) and placed in an ice bath. Glass tubes should not be used since they largely decrease (tenfold less) tlae competence. Usually, 1-5/~1 of the plasmid zeMtion was added to each tube, and the cells were incubated in an ice bath for 30 min. They were then heat-pulsed without agitation at 42°C for 30 s and transferred to an ice bath.
After 0.8 ml of SOC was added, the tubes were placed in a 37 °C incubator and shaken vigorously for 1 h. A desired portion of the mixture was tranferred into a 5 ml polypropylene or glass tube, mixed with 3 ml of melted LB soft agar preincubated at 47°C, and poured on LB plates containing 50 #g Ap/ml. Colonies were counted after overnight incubation at 37 o C



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