Difference between revisions of "Dorman:Selecting T5 resistant E. coli"
(New page: 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 becaus...)
Latest revision as of 01:35, 11 July 2008
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
- The strain from which you'd like to select a T5 resistant mutant
- L-top agar (we can get this from the prep room)
- Phage T5
- L+ plates (Standard L plates with 2.5 mM CaCl2 added immediately before pouring.)
To Isolate phage
- Melt your top agar, then cool to ~50°C in a water bath.
- Aliquot 3mL of to a test tube and place in the heat bath at ~50°C
- Add 100 μl of T5 to the soft agar
- Add 100 μl of overnight grown E. coli, mix, pour onto LB+ plates.
- Grow overnight at 37°C
- 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.
- Pick a colony, transfer to 3mL of L to grow up.
- 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.