Help wanted on TRANSDUCTION
Seyed Mostafa Peighambari
speigham at uoguelph.ca
Fri Apr 7 11:54:17 EST 1995
I am trying to transduce some E. coli strains, one K-12 strian (711) and
One wild type O1 strain (BA22), using P1 phage (clr 100). I have prepared
the P1 lysate on another E. coli K-12 strain (RC73) carrying an Tn10
insertion on cya gene. The titer of lysate is 3 x 1000,000,000.
Essentially, I am following the method of Miller (A short course in
bacterial genetics, p. 263-278. Cold Spring Harbor Lab Press. NY. 1992).
Unfortunately, after many times doing the experiment, no transductant colony
has been isolated. The controls are OK (no colony has been observed in phage
only and bacteria only plates). I use different dilutions of phage from 0,
10 to minus 1, 10 to minus 2,........, 10 to minus 5. 0.1 ml of each
dilution is mixed with 0.1 ml of cells. I have tried both incubations at
30C and 37C for 20 minutes for absorbing stage. I used Citrate buffer, as
Miller recommends, instead of Citrate Sodium but it did not help. I mixed
2ml of 1M Sodium Citrate with 20ml of LB and then I added 0.4 ml of this
mixture to the mixture of phage+cell (after 20 min absorbing stage) and
incubated them for 30 min (and also for 60 min) at 30C (phenotypic
expression). 0.1 ml of above mixture is plated on LB+Tet plates when no
top agar is used or the total volum (0.6 ml) is mixed with 3ml top agar
(LB top agar, which has no glucose. Is glucose important to be included
in top agar?) and is plated. Plates have been incubated in different
temp. 37, 38, 39.5, 40C. LB+Tet plates contain Sodium Citrate and 10x salts
(Miller, 1992). I have also tried plates without NaCitrate and 10x salts.
My mutant strain (RC73, carrying Tn10 on cya) grows well on LB+Tet plate.
Still no transductants????
Any comment is very much appreciated.
Thank you in advance,
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