Help wanted on TRANSDUCTION

[Seyed Mostafa Peighambari]

Hi,
I am trying to transduce an E. coli K-12 strain (711) using P1 phage 
(clr 100). I have prepared the P1 lysate on another E. coli K-12 strain 
(RC73) carrying a 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 Laboratory 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 dilutions is mixed with 0.1 ml of cells. I have tried both
 incubation at 30 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 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 volume (0.6ml) is mixed with 3ml top agar 
and is plated. Plates have been incubated in different temp. 37, 38, 
39.5, and 40C. LB+Tet plates contains Sodium Citrate and 10x salts 
(Miller, 1992). Still no transductants???? 
Any comment is very much appreciated.

Thank you in advance,

Mostafa