Large Volume Marine Cyanophage Phage Purification
Grow up cells in large volumes ( 1 - 2L ) to yield ~ 1011 - 1012 cells using 'microbubbling' technique .
Add Rnase A ( 10ug ml - 1 = for 2L , 250µl of Qiagen's 100mg / ml stock ) and DNase1 ( 0.25 SU ml - 1 = 2L , add 50µl of stock ) .
Centrifuge 12,500 rpm ( 23,975xg ) in Beckman Ja - 14 for 15 minutes at 4°C Filter the supernatant through a Kim Wipe tissue paper .
Mix in polyethylene glycol ( PEG 8,000 ; 100g L - 1 ) by gentle stirring .
Incubate overnight at 4°C .
Collect PEG - phage precipitate by centrifuge ( 6 tubes at a time for the 2L volumes ) at 12,500rpm ( 23,975xg ) in Beckman Ja - 14 for 15 minutes at 4°C .
Discard supernatant .
Gently resuspend pellet into ~ 1 / 150 volume Pro99 ( ~ 1 - 2 ml ) .
Add in resuspension media - place the tube on its side in syrofoam cut - outs so that it rests at an angle .
Put on orbital shaker ( 150 rpm ) in cold room for overnight re - suspension .
Combine resuspended PEG concentrated phage into a single tube ( now ready for layering onto a CsCl gradient ) .
Prepare a CsCl step gradient ( 1.3 , 1.4 , 1.5 , 1.65 bands ) in ultracentrifuge tubes .
Add concentrated phage sample at top .
Spin 28,000 rpm for 4 hours at 4°C in SW28 swinging bucket rotor .
Remove purified phage band ( interface of ρ = 1.4 and 1.5 bands on step gradient ) .
Step dialyze in Slide - A - Lyzer dialysis cassettes ( Pierce # 66425 ; 10K MWCO , 0.5 - 3.0 ml volume ) - 30 minutes against 5xNaCl ( 3M ) , 3xNaCl ( 1.8M ) , two changes of 1xNaCl buffer [ MTM100 = 600 mM NaCl , 100 mM TrisHCL ( pH = 7.5 ) , 100 mM MgCl2 ] .
Store dialyzed particles at 4°C until needed .
Incubate for 1 hour at room temperature .
Add 116g NaCl per Liter of lysate ( thus 2M addition + seawater yields final concentration ~ 2.6M ) .
Incubate on ice 30 minutes .
