Strain included in Kit 7:
||Amp(R), pJS28 specifies P22 gene 9 for making tails|
Strain Reference: Israel et al. (1967). PNAS 57:284
Preparation of P22 Tails:
The following protocol was modified from P. Youderian.
1. Grow PY13579 in 6 liters of LB + amp (50 ug/ml) to overnight density at 37 degrees C. (PY13579 is MC1061 carrying pJS28, which has the gene 9 of p22 for tail protein. It is available from the Salmonella Genetic Stock Centre as SGSC1744.)
2. Pellet cells.
3. To about 12.5 g cell pellet, add 20 ml 0.1M Tris 10mM EDTA, pH 7.5. Transfer to Oak Ridge (40 ml) centrifuge tube.
4. Add 20 mg lysozyme and incubate at 37 degrees C for 1 hour.
5. Incubate at 65 degrees C for 4 hours.
6. Centrifuge: 17K, 70 min., to pellet garbage.
7. Incubate the supernatant at 4 degrees C, overnight.
8. Centrifuge: 17K, 20 min., to pellet garbage.
9. The supernatant should contain crude tails (usually 20 ml at about 10/ml).
Notes -- Some additional garbage may come out of solution, especially if tails are stored over chloroform, but this can be ignored.
Titering and use of Tails:
To determine the titer of a preparation of P22 tail protein, the following method is simple and easy. Starting with a 1:10 dilution of above suspension of tails, make five serial 2-fold dilutions. Mix 0.1 ml of each dilution with 0.2 ml of overnight cells of a P22 sensitive strain and 0.1 ml of a dilution of P22 dmp phage (*) (or of any other tail-defective mutant of P22), sufficient to give about 100 plaques/plate. Add 2.5 ml lambda top agar, spread mixes on lambda plates, and incubate at 37 degrees C overnight. The last dilution at which you see efficient plaque formation (usually around 1:100) is about 10 phage equivalents/ml of tails/plate.)
A Note on Tails and Stocking or Plating Tail Dependent P22:
The following information was prepared by Nick Benson, Sidney Kimmell Cancer Center, San Diego, California:
There are two types of tails, plating tails and stocking tails. Stocking tails are 15x as concentrated as plating tails. The correct dilution of a concentrated tail stock required to make plating tails is experimentally determined as follows.
Mix: 0.1 ml of approximately 1000 tail dependent phage/ml.
0.1 ml of an appropriate bacterial host freshly grown to 4 x 10 cells/ml.
Allow phage and cells to adsorb for 15 minutes at room temperature.
Add 0.1 ml of diluted tails.
Add 2.5 mls of lambda top agar, vortex and pour onto a lambda plate.
Determine the dilution of tails that gave you the largest, most numerous and most uniformly sized plaques. Too few tails will result in tiny or no plaques while too many tails will result in less and widely varied sized plaques. Do not expect a wild-type P22 plaque, since tail dependent phage make small, fuzzy plaques, best seen on lambda plates. Once you have determined the best dilution to use for plating tails you can make stocking tails (15x plating tails). Stocking tails are convenient to use for stocking a tail-dependent phage.
Stocking a Tail Dependent Phage:
1. Mix: 3 mls of a fresh overnight of the bacterial host.
27 mls of LB
0.05 mls of stocking tails
One fresh plaque grown on the same host
2. Shake overnight at 30 degrees C or 37 degrees C.
3. Add 1 ml of CHCl3 and shake vigorously. Allow the mixture to settle and decant the LB.
4. Spin cell debris down in the Sorval, 8K, for 10 minutes.
5. Add the LB to a new tube and add 0.05 mls of stocking tails. Allow the lysate to incubate at 37 degrees C for 2 hours or at room temperature overnight.
6. Spin the tailed phage down, 16K, for 1 hour. Discard the supernatant and resuspend the pellet in 5 mls of buffered saline. Add 0.2 mls of CHCl3, vortex and store the stock at 4 degrees C.
Note -- the above procedure is a simple and practical method for the successful use of a concentrated stock of gene 9 (tail) protein. The concentrations and amounts of tail used above are arbitrary but useful. A more detailed explanation on tail protein and "tail equivalents" can be found in PNAS 57:284, 1967.