Seyed Mostafa Peighambari (speigham at uoguelph.ca) wrote:
: 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????
Your recipient strain (711) isn't recA- is it? That would explain your
problems as the incoming DNA would not recombine into the chromosome.
Vince Mulholland
