Before Density Gradient Centrifugation can begin, make sure you have:

 

1.    Phage concentrate (either via spin filters or via pelleting and resuspension) in about 30 ml of solution (or 15 ml, if possible).  Each CsCl gradient tube can accommodate 15 ml phage concentrate to be purified.

2.    Sterile (autoclaved) CsCl solutions made up in SM buffer:

1.70 g/ml:    95g CsCl (s) +  75 ml SM

1.50 g/ml:    67g CsCl (s) +  82 ml SM

1.45 g/ml:     60g CsCl (s) +  85 ml SM

 

Preparation of density gradients:

 

1.    Add 0.5 g CsCl (s) per ml of phage suspension.  Invert gently until completely dissolved.

2.    Carefully pipette CsCl layers into a clean Beckman SW 28 polyallomer tube.  1.70 g/ml on the bottom, then 1.50 g/ml, and finally 1.45 g/ml (see Figure 1).  Pipette slowly to minimize mixing of layers.  As each layer is completed, mark it on the outside of the tube with a permanent marker so that the interfaces can be identified at the end of the centrifuge run.

3.    Carefully pipette15 ml of the phage suspension (1.15 g/ml with CsCl added) on top of the CsCl layers in the tube.  

4.    Place the tubes in SW 28 buckets and load the buckets on to the SW 28 rotor.  Tubes should be spun at 22 000 rpm at 4 degrees C for a minimum of 2 hours, but can be spun overnight if desired.

5.    After the centrifuge run is complete, use a 25 ml pipette to aspirate the upper portion of liquid in the tube; to ensure that all phage are recovered, collect all liquid to just below the 1.45 g/ml – 1.50 g/ml interface.  Store the purified phage concentrate in a sterile 50 ml centrifuge tube.

6.    Use a Millipore Centricon 20 spin filter to reduce the volume of recovered CsCl purified phage concentrate (this will take several passes).  Balance your sample tube with a used centricon tube filled with an equal volume of water.  Make sure to rebalance tubes after each centrifuge run.  Set the centrifuge to 4000 X g, 4 degrees C, and begin spinning for 4 minutes.  Adjust spin times as necessary to pull most of the liquid through the filter, but DO NOT spin the filter dry.  

7.    After the volume has been reduced, wash the concentrate with three volumes of sterile SM buffer to remove Cs from solution.  After the third wash, pour off the small volume of purified phage concentrate into a sterile 15 ml centrifuge tube (should be 1-3 ml).

8.    (See Centricon instructions for more details) Invert the filter in its receiving cup and spin at 1000 X g for 5 min.  Use a pipette to transfer the recovered liquid to the 15 ml centrifuge tube.  Store concentrate at 4 degrees C until use.