Method: Liquid Phage Lysates

June 7 1990

Srini Ramachandra

Purpose:

5 ml liquid lysates are prepared when a small amount of DNA from a large number of lambda clones is needed. The lysates can be made using 10- 20 ul of a stock lysate or a 100-fold amplified phage "macroplaque" as the inoculum.

Time required:

2 days

Day 1
• Macroplaque preparation:

  1. Overlay an LBM plate containing 1mM MgCl2 with 0.1 ml of a fresh overnight cell culture LE392 in 3 ml LBM top agar. Let solidify at room temperature.
  2. Transfer phage from a single well-isolated plaque to its grid position using a sterile toothpick or wood applicator stick. Inoculate an area about the size of the wide end of a pasteur pipet by lightly touching the agar surface several times.
  3. Incubate at 37deg.C. The grid areas should clear in 6-7 hours creating macroplaques. (Avoid overnight incubation).
  4. Chill plates at least 30 minutes.
  5. Excise macroplaques using the wide end of sterile pasteur pipets attached to a syringe in a plug-pulling assembly or to a pi-pump.
  6. Put each plug into a 15 ml disposable tube containing 250 ul lambda diluent. Phage extraction of the agar plug can be done at 4 degrees C overnight or by gently shaking for 1-2 hours. At this point phage plugs can be stored at 4 degrees C for several weeks provided they are tightly capped.
  7. Inoculate 5 ml LBM medium with LE392 and grow overnight at 37 degrees C.

Day 2
• Lysate preparation:
  1. Add 300 ul of a fresh undiluted overnight culture of LE392 to the tube containing the macroplaque (from step #6 above). Incubate 15 minutes at 37 degrees C or 20 minutes at room temperature.<
  2. Add 5 ml LBM containing 1mM MgCl2. (Correct Mg concentration is important!). Also prepare 2 controls: one with cells + LBM and one with lambda diluent + LBM.
  3. Place tubes on a roller drum in a 37 degrees C incubator. Check in 2 hours to see that cultures are growing. When lysates clear remove from incubator and store at 4 degrees C until ready to proceed (lysates should clear in 4 hours at most 6 hours).
  4. Reduce viscosity of cleared lysates by adding 50 ng DNaseI (50 ul of a fresh 1:1000 dilution of a 1 mg/ml stock. DNaseI stock is prepared in 0.15M NaCl and is stored in 50% glycerol at -20 degrees C). Incubate at 37 degrees C for 30 minutes.
  5. Add one half volume 10mM Tris-HCl to the lysate. Spin at 2500 rpm for 30 minutes to remove the cell debris. Decant the supernatant into a clean tube. For a stock of the lysate remove 0.5 ml to an eppendorf tube add a drop of chloroform and store at 4 degrees C.

     The remainder of the lysate can be stored at 4 degrees C (for at least 1 week) until needed for the DNA prep protocol.

Helms C. Dutchik J.E. and M.V. Olson. (1987). "A lambda DNA protocol based on purification of phage on DEAE cellulose". In: Meth. Enzymol. 153: p 69-82. Eds.: R. Wu L. Grossman. Academic Press.