THE JOHNS HOPKINS UNIVERSITY
  SCHOOL OF .HYGIENE AND PUBLIC HEALTH
        e*s N0RTl-l WOLFE STREET

March 4, 1958

Dr. Joshua Lederberg
Department of Medical Genetics
Genetics Building
University of Wisconsin
Madison 6, Wisconsin

Dear Joshua:

  The problem of growing competent cells in media other than Levinthal
broth has so far been very disappointing.  After some intensive original
experiments we used a modified medium which I described in a previous
letter, and except for an occassional foray have continued to use this
medium for preparing competent cells.

The procedure we use is as follows:

  (1) Inoculate approximately 0.5 ml. of fully grown culture into
50 ml. of Levinthal broth (1 part Levinthal stock and 1 part Eugonbroth).

  (2) Grow cells with aeration to a concentration of lo9 cells
perml.  On our Coleman Junior at 650 mu with an adapter for 10 x 100 mm.
pyrex tubes this corresponds to a turbidity reading of .09 (0.D).

  (3) The cell suspension is then transferred to a large test tube
or small flask whfch is kept at 36037oC. for an additional 90 minutes.
Some growth does occur usually to about 1.5 x loo/ml. Cells grown
aerobically would grow to about 6 x loo/ml. in this period of time.

  The method of preparing competent cells a la Alexander and Leidy
consists of growing the cells until they &mes% approach saturation.
We reasoned that the competency might be more related to aeration or growth
limitation than to cell number; conseque&lyj we restricted the aeration
of the culture during early log phase growth and found the cells to be
competent.  Actually the culture may be grown to any log phase concentration,
placed under non-aerobic conditions, and the cells will become competent.

  With regard to the stability of DNA, we have kept preparations in
the refrigerator for almost three years (in fact, the first preparation
I ever made) and the potency of this DNA is as good as ever.


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  It does not matter when the DNA is added to the cells as long as
they are competent for some period during exposure. It is important,
however, that there be enough free divalent cations to give about
.002 M excess over any chelating agents such as citrate which might
be used to stabilize the DNA.  We generally use Mg+t for this purpose;
Ca++ is also O.K.  Ca++ would be better in the case of pneurococcus
transformations since there appears to some DNAse liberated by Pn.

   I am forwardin  sac Levinthal stock which should be diluted ltl
with Eugonbroth,  an b "i'sample of DNA.  If for an;y reason you have
difficulty in preparing competent cells, I can ship you some of ours
packed in dry ice.  In this case it will be necessary to coordinate
air freight schedules so that someone can pick up the package as
soon as it arrives.

  If it is convenient, would you please try to reserve space in the
dormitory for me for the period of the symposium.
for dormitory space,                  If you are crowded
          it would suit me quite well to have whatever hotel
accommodations are available.  I em planning to give a course in Genetics
next fall to people in the Medical Institutions, and I would appreciate
the opportunity to obtain a closer look at some of the areas under
discussion.

  If you have any difficulties whatsoever, please do not hesitate
to write.

Sincerely,

Sol H. Goodgal

SHGrcsb