Ultracompetent E.coli cells?

Paul N Hengen pnh at cockleberry.ncifcrf.gov
Tue Dec 5 13:10:04 EST 1995


Stephan Witte (stephan.witte at uni-konstanz.de) wrote:

: I'm looking for highly competent E.coli cells for a library transformation.
: Our homemade CaCl2-competent  cells yield app. 5E4  transformants per ug
: DNA (6 kb plasmid)
: I think, this is not much.

If you meant 5x10^4 per microgram DNA, you are right. That is very low.

: Are there better protocols for preparing highly competent coli-cells?

To begin with, pick a good strain of E. coli. What are you currently using?
There are a number of references on the FAQ list for this group under the
question about transformation. If you are constructing a library to find
rare DNAs, you might consider electroporation.

: What to think about Stratagenes ultracompetent cells? What's the trick?

I don't know about Stratagene, but Epicentre uses a modified version of the
Chung technique which should yield up to 1x10^8 transformants per microgram.

@article{Chung1989,
author = "C. T. Chung
     and S. L. Niemela
     and R. H. Miller",
title = "One--step preparation of competent {{\em Escherichia coli}}:
transformation and storage of bacterial cells in the same solution",
journal = "Proc. Natl. Acad. Sci. USA",
volume = "86",
pages = "2172-2175",
month = "apr",
comment = "also see Epicentre Technologies Forum vol.2, no.3, pg.5;
chemical transformation of bacteria",
year = "1989"}

ABSTRACT:

| We have developed a simple, one-step procedure for the preparation of
| competent Escherichia coli that uses a transformation and storage
| solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene
| glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5].
| Cells are mixed with an equal volume of ice-cold 2 x TSS and are
| immediately ready for use. Genetic transformation is equally simple:
| plasmid DNA is added and the cells are incubated for 5-60 min at 4
| degrees C. A heat pulse is not necessary and the incubation time at 4
| degrees C is not crucial, so there are no critical timing steps in the
| transformation procedure. Transformed bacteria are grown and selected by
| standard methods. Thus, this procedure eliminates the centrifugation,
| washing, and long-term incubation steps of current methods. Although
| cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest
| transformation efficiencies (10(7)-10(8) transformants per micrograms of
| plasmid DNA), cells harvested at other stages in the growth cycle
| (including stationary phase) are capable of undergoing transformation
| (10(5)-10(7) transformants per micrograms of DNA). For long-term storage
| of competent cells, bacteria can be frozen in TSS without addition of
| other components. Our procedure represents a simple and convenient
| method for the preparation, transformation, and storage of competent  
| bacterial cells.

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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              /--------------------------/*
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