IUBio

P1 transduction of Cm marker

Rafael Maldonado rafamaldo at geocities.com
Tue Mar 23 05:50:28 EST 1999


Stephan Heeb wrote:

> Before modifying this protocol and attempting again, I would like to know
> if there's someone here that has some PRACTICAL EXPERIENCE with this
> transduction that could give me some advice.

It must be hundreds of people with practical experience in transduction
with P1. All of them will tell you that the first experience with P1 may
be frustrating. Moreover, if you have worked before with other "easier"
phages, as lambda or P22, even more frustrating it is.

First, I recommend to follow the Miller (1992) protocol without
modification. It has been proved to work very well, and there is no
reason to modify it. Unless you have a very special reason, which I
don't know. In case this does not work, you may now try to modify it.


>   -After preadsorbtion of the phage and addition of 1M sodium citrate, 0.6
> ml of  MME medium is added (final vol. 1.0 ml). The cells are then
> incubated for 1 hour at 37°C with shaking, and then plated on MME plates
> containing 20 ug/ml Cm.

As Miller says, sometimes is better use 0.2 ml od 0.1 M Citrate,
particularly for antibiotic resistance markers. About MME, I supose it
is minimal medium E. I would stick to LB with A salts and 5x10E-3 sodium
citrate. I've never used minimal medium for transduction, and unless
there is a very good reason to use it, LB is you better choice. I have
found that expression time for CmR is not necessary. You may plate
directly on LB+salts+citrate plates with Cm. Of course, expression time
will not be bad, unless that during that time the phage is killing your
bacteria.
 
> The aim of the extra incubation time is to allow for the recombinations to
> happen before plating on selective media. 

The main reason for that extra incubation time is expression, not
recombination, of the resistance gene. Recombination is almost
instantaneous; but there must be time for expression of the gene of
resistance. If you fells that expression time is needed, you should do
it in LB+salts+citrate.

> MME medium is used, since it
> contains citrate to chelate calcium. We also don't want a lysogenic
> result.

That depends mainly in which kind of P1 you have. P1 vir almost never
lysogenize cells, so you are safe. The problem is that P1 vir kills all
your cells, even the transductans. The citrate is there to advoid P1 to
propagate, it chelates the Ca ions which the phage needs. It does not
advoid the lysogenic pathway, in case there was one. As Miller says,
"allowing time for expression after transduction with a virulent phage
is tricky, since the phage can reinfect and kill potential
transductants". So plate directly, and see what happens.
 
I hope this helps. If you have additional doubts, please contact again.

-- 
Rafael Maldonado
Division of Genetics
University of Alicante, Spain
rmaldonado at ua.es



More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net