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 .
