Help wanted on TRANSDUCTION
Seyed Mostafa Peighambari
speigham at uoguelph.ca
Wed Apr 5 21:10:38 EST 1995
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.
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,
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