Method: Phage Plate Stock Lysates

June 7 1990

Srini Ramachandra

Purpose:

To prepare phage lysates to be used for small or large scale phage DNA preps. This method usually produces lysates with titers of 2-8x10e10 pfu/ml.

Time required:

2 days

Procedure:

Day 1

1. Prepare LBM plates and LBM top agar. Also prepare an LE 392 overnight culture at 37deg.C in LBM containing 0.2% maltose and 1mM MgSO4. Plates should be freshly prepared (not more than one day old).

Plating the phage:

1. Mix 0.1 ml overnight culture with 10e6 plaque forming units (pfu) lambda phage. (Use 2-4 x 10e5 pfu phage when using wild type lambda or large plaque-former). Also prepare one tube with 0.1 ml overnight culture and one tube with 0.1 ml lambda diluent as controls.
2. Incubate at 37deg.C for 15 minutes.
3. Add 3 ml LBM top agar (50-55deg.C) mix briefly pour and swirl the mixture onto a fresh LBM agar plate.
4. Allow top agar to set for 5-15 minutes on benchtop. Put plates media side down into a plastic box containing very wet paper towels on the bottom. Put on a plastic lid and incubate at 37deg.C for 6 to 6.5 hours.

   Little if anything can be seen on the plates before 5 hours. The best time to harvest the plates is when nearly confluent lysis occurs. When nearly confluent lysis has occurred chill plates briefly.

Harvesting the phage:

1. Add 5 ml lambda diluent to the surface of each plate and chill them at 4 degrees C overnight (2-3 hours is probably enough time).

Day 3

1. Remove the lambda diluent (with phage) to a sterile tube. Keep the lysate at 4 degrees C when possible from this point onwards. Double the volume with TE or 10 mM Tris-HCl and spin at 2500 rpm to remove the cell debris. To prepare a stock of the lysate remove 0.5 ml add 1 drop of chloroform mix and place at 4 degrees C for storage.
2. Store the remaining supernatant in a clean sterile capped tube at 4 degrees C until ready to purify phage.

References: see liquid lysate procedures