Help wanted on TRANSDUCTION

Mr V Mulholland lsrdp at csv.warwick.ac.uk
Thu Apr 13 02:50:15 EST 1995


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



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