Dorman:Selecting T5 resistant E. coli

Resistance to the bacteriophage T5 is a useful genetic marker for competition experiments with E. coli because it has no effect on fitness (typically- you'll want to check this) and because it is easy to select for.

What you'll need

 * 1) The strain from which you'd like to select a T5 resistant mutant
 * 2) L-top agar (we can get this from the prep room)
 * 3) Phage T5
 * 4) L+ plates (Standard L plates with 2.5 mM CaCl2 added immediately before pouring.)

To Isolate phage

 * 1) Melt your top agar, then cool to ~50°C in a water bath.
 * 2)  Aliquot 3mL of  to a test tube and place in the heat bath at ~50°C
 * 3) Add 100 μl  of T5 to the soft agar
 * 4) Add 100 μl of overnight grown E. coli, mix, pour onto LB+ plates.
 * 5) Grow overnight at 37°C
 * 6) Pick several colonies from the agar and streak them out on L. You'll probably find that there are several different colony phenotypes on the plates- avoid the excessively mucoid colonies.
 * 7) Pick a colony, transfer to 3mL of L to grow up.
 * 8) Before you put in in the incubator, test to confirm T5R: streak T5 across an LB/Ca plage, then streak the strain(s) of interest perpendicular to this.  You should see a break at the streak for T5S, but no break for T5R.