Cesium Chloride Gradients
Prepare cesium chloride densities of p1.2 , p1.4 , p1.5 and p1.65 in seawater ( sw ) that has been 0.02μm ( or 0.2μm ) filtered and autoclaved .
Place tube of prepared cesium on balance .
Tare balance to 0 .
Remove 1ml with sterile pipet tip .
Layer CsCl from the bottom using a gradient fractionator if you have one or by hand .
Carefully layer on sample ( ~ 5ml can be layered with SW40 ~ 15ml with SW28 ) .
Weigh and balance tubes .
Centrifuge using SW40 or SW28 Beckman rotor @ 24,000rpm for 4hr at 4°C .
Have sterile 0.5ml tubes ( SW40 ) or 1.5ml tubes ( SW28 ) labeled 1 - 24 Open tubes in a rack .
Place tube on ring stand .
Puncture about 2mm from the bottom side of the tube using a sterile 20 guage needle , bevel up .
Collect droplets to fill each tube in order .
Place tube with sample on 4 - place balance in holder .
Tare to 0 .
With sterile pipet tip , remove 100µl .
Take measurement .
Return sample to tube .
Remove another 100µl , take measurement , return sample to tube .
Remove yet another 100µl , take measurement , and return sample to tube .
After all tubes have been measured , take average of the 3 values for each tube .
Multiply by 10 .
Pool and extract DNA or can confirm presence of virus first by staining 10µl with SYBR gold .
Count under fluorescent scope .
Pool all samples with virus .
