Help wanted on TRANSDUCTION

[Seyed Mostafa Peighambari]

Hi, 
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,

Mostafa