CsCl Step Gradient to Purify Phage Apr. 2000 chp

Phage are best purified by a CsCl gradient. This is separated by density not sedimentation as in a sucrose gradient. This is a protocol for P22 like phage not T4 with delicate tail fibers.

1) Make your phage, I am used to being at about 1E9-1E10 /ml. Now it needs to be concentrated. To put on the top of the gradient we use two methods:

a. In the Beckman Ti45 1 hr 35K

b. PEG ppt using 10% PEG (6K) 0.5 M NaCl (Yamamoto 1970 Virology 40, 734-744)

1) Cammie adds it dry
2) Patricia adds it as an autoclaved 50% weight/ volume.

It is added, mixing in the cold and allowed to sit at least 1 hr but best over night.

2) The phage is collected by cfg, 8K GSA 20 min a waxy pellet resuspend in a small volume. Keep in mind you will need to load on to a gradient if using a SW 28.1 about 3-5mls /tube. If SW50.1 about 0.5-1 mls.

3) Prepare CsCl step gradient. [Cammie's recipe unknown source]

M = 8 (p25-1) MW = 168.37

I weigh out the CsCl, add a little buffer with the stir bar stirring, adding CsCl slowly till all the CsCl is dissolved. I bring up the volume, rinse the stir bar. The buffer should be high in Mg²⁺ to stabilize the heads.

50 mM Tris pH 7.6
100 mM MgCl₂

Grams of CsCl to be added to Buffer

p	M	25 mls	50 mls	75 mls	100 mls	200 mls
1.3	2.4	10.10	20.20	30.31	40.41	80.82
1.4	3.2	13.47	26.94	44.16	53.86	107.76
1.45	3.6		30.24		60.48
1.5	4	16.87	33.74	50.61	67.48
1.55	4.4	18.48	36.96		73.92
1.6	4.8	20.2	40.40
1.65	5.2	21.89	43.78		87.55
1.7	5.6	23.57	47.15	70.7	94.29

4) Make gradient in a SW 28.1 Beckman ultra clear tube holding 17 mls. I like them since you can see; the polyallomar are easier to puncture but you can't see through them.

I layer the lowest density first, then I displace it with the next heavier using a long canular needle and displace with the next heavier and so on. I slow layer sample on top. I do not want the sample volume to be more than half the gradient I try to keep it to less than a third. Below are the volumes I use. If you can, mark the layers.

1.3 is 4 mls, 1.4 is 4 mls, 1.5 is 3 mls and 1.7is 2 mls

5) Centrifuge for 2.5 hr 24K in the SW 28.1; if run longer the bands will stay at the same position since it is an equilibrium density gradient.

6) Draw a sketch of the layers in the tube.

7) Phage are a bluish whitish band is between the 1.4 and 1.5. The higher bands are empty heads and the bottom stuff are ribosomes. The band between 1.4.-1.5 can be pulled out with a syringe and 20 or above gauge needle. Put a bit of stop cork grease on the tube and the middle of the needle, this will act as your sealant .

a) Plunge the syringe a few times, if you don't, it is tough to start pulling the band. Puncture by slowly twisting and pushing the needle, beveled side up, a few mm below the bluish white band.

[Note: once through, the resistance decreases and you might go through the other side so slow and even pressure is the key.]

b) Now through ensure the hole around the needle is sealed with grease. Collect the band by slowly moving the needle back and forth under the band.

c) Change needle to a 18 gauge if using in a Pierce Dialysizer, if not, take needle off and put in a tube till you can dialyze it .

Change needle and syringe before going to the next tube even if it is the same sample. Repuncturing the tube dulls the needle enough it can cause problem on the next collection.

8) Dialyze the band against a high Mg buffer. T4 needs to have step dialysis 1.1 because the heads will pop if put directly into a buffer without CsCl.