about transformation of DNA into E.Coli

Paul N Hengen pnh at ncifcrf.gov
Thu Aug 8 15:28:33 EST 1996


Paul N. Hengen (pnh at ncifcrf.gov) wrote:

| 1. Pre warm the plates @37 (very imp) for 30min or 60min which ever is
| convient to you.
| ...
| 9. Take the entire amount and spread on the prewarmed LB +antibiotic
| plates (no SOC etc etc).

> In my opinion, you are still heat shocking your cells by plating on
> pre-warmed media. I've seen people heat shock at 37C for 10 min. before
> plating and they swear it works better than what I normally do, which
> is 42C for 90 sec. At one time I did a side-by-side comparison and
> didn't see any difference, so I never switched. I'd really like to see
> your numbers of transformants per ug DNA compared with other times
> and temperature of heat shock if you have any.

Sailesh:

I've now found the section of the Chung1989 paper which describes their
protocol using TSS for making JM109 competent cells and doing transformation
without a heat shock.  They write on page 2174 that using 100 pg of plasmid DNA
per 100 ul of competent cells the efficiency was 5.35 +/- 0.43 x 10^7
transformants per ug DNA when incubating the mixture on ice for 5 min. and 2.38
+/- 1.17 x 10^8 when incubating the mixture on ice for 30 min.  They say that a
heat shock is not necessary for their procedure.  Still, the reported numbers
are 100-1000 fold less than those reported elsewhere for electro-transformation
which is 1 x 10^10 transformants per ug DNA. How does this compare with your
results?

This is different than for cells made competent by the Hanahan procedure in
which he optimized the heat shock. Hanahan writes a little bit about this in a
book chapter (see below), but right now I can't find where he did the complete
study. I remember seeing it somewhere, perhaps in his thesis, which I no longer
have a copy of.  Where did the original heat shock idea come from anyway? Cohen
heat shocked his cells at 42C for 2 min., which was published in 1972, but
maybe this came from work done with naturally transforming bacteria (?).

@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",
year = "1989"}

@inbook{Hanahan1985,
author = "D. Hanahan",
editor = "D.  M. Glover",
title = "{DNA} cloning: A practical approach",
chapter = "Techniques for transformation of {{\em E. coli}}",
publisher = "IRL Press",
volume = "I",
address = "Oxford",
pages = "109-135",
year = "1985"}

@article{Cohen1972,
author = "S. N. Cohen
     and A. C. Y. Chang
     and L. Hsu",
title = "Nonchromosomal antibiotic resistance in bacteria:
genetic transformation of {{\em Escherichia coli}} by {R-factor} {DNA}",
journal = "Proc. Natl. Acad. Sci. USA",
volume = "69",
pages = "2110-2114",
year = "1972"}

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