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 .
