Parashar Lab

Making SPP1 Phage Lysates

 

Day 1

End of the day streak out desired strain

 

Day 2- Prepare Lysate

Grow 3 mLs of your desired strain in TY broth.

 

Prepare a dilution of the SPP1 phage in TY broth (Diluting from -2 to -6 should be enough.)

 

Take the -4, -5, and -6 dilutions and put 100 ul of each into clean tubes.  This is another dilution step, so these three samples are now the -5, -6, and -7 dilutions.

 

Add 200 ul of cells to each of the final three dilutions.  Incubate statically at 37ºC for 15 minutes.

 

While the cells are incubating, remove your fresh TY agar plates from 4C and heat up your TY soft agar bottle.

 

Using disposable pipets, put 3mL of TY soft agar into each dilution tube.  Working quickly, vortex your sample and dump the entire contents of the tube onto a TY plate.  (You don’t want the molten agar to solidify on the plate just yet!) Swirl the plate around so that the mixture covers the entire plate surface.  Repeat for each dilution.

 

Let the plate dry on your bench for 5 minutes and then dry the plates in the hood for 10-15 minutes.  Incubate O/N at RT or 30C but do not invert the plate.

 

Day 3- Harvest Lysate-

 

You will need 2x15mL conical tubes per lysate.

 

Add 5mL of TY broth to the dilution plate that is best for plaques.  (Examine plates for lysis.  Select the plate that has so many plaques that you can only see ridges of cells from the lawn.)

 

Using an ethanol flamed hockey stick, scrape up the soft agar (gently).  Be sure to scrape all around the edges of the plate.  Use the hockey stick to break up the agar into smaller pieces.

 

Dump the scraped up soft agar and TY broth into a 15mL conical tube.  Add 10ul of DNase 25ug/ml to each harvested lysate.  Vortex thoroughly to release the phage from the agar.  Spin for 10 minutes at 5800 rpm.

 

Filter lysate using 0.45 µm syringe filter into a new 15 mL conical tube.  Label and refrigerate at 4C.

Transduction

-Inoculate recipient in 3 ml TY broth. Grow at 37ºC until culture is very dense.

-Mix 1 ml of culture with 25 µl of phage stock in 15 ml conical tube. (For mls and tet, use 10 ul)

-Add 9 ml TY to conical tube and incubate 30 min statically at 37ºC.

-Centrifuge 5000g for 10 minutes.

-Pour off supernatant.

-Resuspend pellet in remaining volume.

-Plate all of sample on selective media + 10mM citrate. (It is not necessary to plate tet transductions on citrate.)

-Incubate 37ºC overnight.

 

 

More notes:

Fresh media is critical. Do not dry plates. 

If there are too many plaques, some sort of lysate parent contaminant/phage resistant colony grows on the top agar.  Try to avoid them.

Do not incubate transduction step more than 30 minutes, because phage burst can occur after 45 minutes, causing decrease in OD and lessen chances for successful transduction.

Citrate blocks phage superinfection killing and increases the transduction frequency by perhaps 100 fold.  Citrate is absolutely essential for obtaining transductants using the chloramphenicol resistance marker.  Citrate also improves spec and kan transduction.  In tetracycline transductions the citrate seems to inhibit transduction.

It may take as long as 24 hours to get colonies in mls or tet selections.

Drying plates after pouring on top agar seems to help make nicer plaques.