Isolation and Purification of DNA from Chlorella Viruses
For each gradient, in 100 x 13 mm tubes, mix together 500 µL of virus, 60 µL of 10X TEN, pH 7.4, and 60 µL of 1% Na sarcosyl.
Add 600 µL of 60% (w/w) CsCl to each tube.
Heat the tubes at 75°C for 15 min.
Layer the samples onto pre-formed 40-60% (w/w) CsCl gradients in SW60 rotor tubes (to make a final 30-60% gradient).
Add 1200 µL of the heated virus to each gradient.
Centrifuge the gradients in SW60 rotors at 35,000 rpm, 18 hours, 25°C.
Remove the DNA bands from the gradients with a wide bent needle to silicon-coated 30 mL corex tubes.
Extract the Hoechst dye from the DNA by adding an equal volume of CsCl/TE-saturated isopropanol to the DNA solution, mixing gently by inversion, centrifuge for 1 min at 3,000 rpm in the Sorvall to separate the phases, and pipet off the upper isopropanol layer.
Repeat last step.
Add 1.0 mL of 3 M NaOAc to each tube and adjust the volume of each tube to 10.0 mL with 1X TE buffer.
Precipitate the DNAs with 2X volumes of 100% EtOH.
Mix well and hold at -20°C overnight.
Centrifuge the DNA samples in the Sorvall HB-4 rotor at 10,000 rpm, 20 min, 4°C.
Discard the supernatants.
Dry the pellets briefly (10-15 min) in the vacuum desiccator to remove the EtOH.
Resuspend the DNA samples with approximately 500 µL of 1X TE buffer.
Determine the DNA concentrations and store the DNAs at 4°C.
