Could anybody share a protocol of using CsCl-gradient ultracentrifigation for phage purification with details. It's a new isolated phage, so how should I choose the gradient density to try first? And if I will use the product to inject animals, what kinds of cleaning procedure can I use?
Look at Jose A Sivera-Marza profile for a reference. Using a step CsCl gradient (1.7, 1.5, 1.3) is usually convenient .
Make a CsCl stock solution of 62.5%. Make four gradients as follows 1) CsCl/TE 1:0 2) CsCl/TE - 2:1 3) CsCl/TE 1:1 3) CsCl/TE 1:2. USe Ultracentrifuge and add this gradient slowly in the ultracentrifuge tube 0.2 ml , 1.3 ml, 1.3ml and 0.2ml. Finally add the Phage slowly 1.3ml . Centrifuge the tubes 40 min at 40,000 rpm. Output: Top 1/2 layer of empty phage 2/1 phage particles and lower turbid band of carbohydrates.
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