CsCl Step Gradient to Purify Phage
Make your phage: author used to being at about 109-1010 /ml.
To put on the top of the gradient we use two methods.
First, In the Beckman Ti45 1 hr 35K and second, PEG ppt using 10% PEG (6K) 0.5 M NaCl (Yamamoto 1970 Virology 40, 734-744).
Mix in the cold and allow to sit at least 1 hour; recommended overnight The phage is collected by cfg, 8K GSA 20 min a waxy pellet resuspend in a small volume.
Prepare CsCl step gradient.
[Cammie's recipe unknown source]  Make gradient in a SW 28.1 Beckman ultra clear tube holding 17 mls.
Layer the lowest density first Displace it with the next heavier using a long canular needle.
Displace with the next heavier, and so on.
Slow layer sample on top.
Centrifuge for 2.5 hours 24K in the SW 28.1.
Draw a sketch of the layers in the tube.
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.
Plunge the syringe a few times.
Puncture by slowly twisting and pushing the needle, beveled side up, a few mm below the bluish white band.
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.
Change needle to a 18 gauge if using in a Pierce Dialysizer, if not, take needle off and put in a tube until you can dialyze it.
Dialyze the band against a high Mg buffer.
