Cesium Chloride Protocol for Phage
Filter cesium solutions with 0.02µm filter .
Mark top of first gradient layer with pen before adding next layers .
Set up gradient as ( from top to bottom ) : 1ml of 1.7 g / ml , mark top of layer 1ml of 1.5 g / ml , mark top of layer 1ml of 1.35 g / ml 8.5ml of 1.15 g / ml - this is phage concentrate .
Add CsCl to your sample .
Check balance of tubes .
Centrifuge at 22,000 rpm for 2 hours at 4°C , approximately 60 - 80,000xg .
Pierce tube at 1.7 / 1.5 g / ml interface .
Bevel up , and collect 1.5 mls ( should include 1.5 g / ml step and 1.5 to 1.35 interface ) .
Check with slides for virus particles , etc .
Use 20 µl from [ viral ] fraction and 500 µl from " upper " layers .
Store in fridge at 4°C until ready to extract .
Add 0.2 vol chloroform , mix and incubate 10 min . at room temperature .
Spin at 4000 rpm in table Beckman for 10 minutes to separate phases .
Save and transfer top phase to new 15 ml tubes .
Add 10 - 15 µl DNase I ( 1 mg / ml in H2O ) to phage sample ( 1.2 - 1.5ml ) .
Incubate 15 minutes at 37°C .
Treat with RNase if RNA is to be extracted .
Inactivate for 15 minutes at 65°C .
Transfer all to new " oak ridge " tube for faster centrifugation later .
Add 0.1 volume 2 M TrisHCL ( pH 8.5 ) / 0.2 M EDTA .
Add 100 µl 0.5 M EDTA per 10ml .
Add 1 volume of formamide .
Add 10 Fl glycogen Incubate at room temperature for 30 minutes .
Add 2 volumes of room temperature 100 % ethanol .
Pellet in Sorvall ( 12,000 rpm for 20 minutes ) .
Wash with 70 % ethanol , two times .
Resuspend into 567 µl TE and continue with CTAB extraction .
Resuspend pellet in 567 µl TE .
Add 30 µl of 10 % SDS and 3 µl of 20 µg / ml proteinase K .
Mix .
Incubate 1 hour at 37°C - 56°C .
Add 100 µl of 5 M NaCl and mix thoroughly .
Add 80 µl CTAB / NaCl solution .
Mix .
Incubate 10 minutes at 65°C .
Add equal volume of chloroform ; mix .
Microcentrifuge for 2 minutes .
Transfer supernatant to separate tube .
Add equal volume of phenol / chloroform to the supernatant fraction ; mix .
Microcentrifuge for 2 minutes .
Transfer supernatant to separate tube .
Add equal volume of chloroform to the supernatant fraction ; mix .
Microcentrifuge for 2 minutes .
Transfer supernatant to separate tube .
Add 0.7 volume isopropanol to the supernatant fraction .
Mix gently until DNA precipitates .
Centrifuge 15 minutes in cold .
Wash with 70 % ethanol .
Resuspend in 50 µl Sigma water Check O . D .
using the Nanodrop
