Chlorovirus DNA Miniprep Procedure
Infect appropriate host with desired Chlorovirus species at moi of ~ 0.001 - 0.01 .
Incubate the samples at 25°C for 24 - 72 hours , with continuous light and shaking .
Centrifuge 30 mL of the lysates in the Sorvall SS34 rotor at 5,000 rpm ( 3,000 rcf ) , 5 min , 4°C .
Add 10 % NP - 40 ( or Triton X - 100 ) to the lysate supernatants for a final concentration of 1 % .
Centrifuge the material in a Beckman Ti 50.2 rotor at 15,000 rpm ( ~ 27,000 rcfmax ) , 75 min , 4°C .
Resuspend the virus pellets with 1.0 mL of 50 mM Tris - HCl , pH 7.5 , 10 mM MgCl2 .
Transfer 500 µL of the resuspended virus to 1.5 ml screw - cap microfuge tubes .
Add 9 µL of DNAse I and mix .
Add 6.0 µL of 500 mM EDTA , pH 8.0 to the samples and mix .
Add 300 µL of buffer - saturated phenol and 300 µL of CHCl3 : Isoamyl alcohol ( 24 : 1 ) to the tubes .
Centrifuge in the microfuge at maximum speed for 5 min at 4°C .
Remove the upper aqueous layers to clean tubes .
Add 600 µL of CHCl3 : Isoamyl alcohol ( 24 : 1 ) to the tubes .
Remove the upper aqueous layers to clean tubes and repeat the CHCl3 : Isoamyl alcohol extraction 1X .
Add 66 µL of 3 M NaOAc to each tube .
Precipitate the DNAs with 2X volumes ( approximately 1350 µL ) of 100 % EtOH .
Centrifuge the tubes in the microfuge for 10 - 15 min at 4°C to pellet the DNAs .
Wash the DNA pellets 1X with 1000 µL of 70 % EtOH in the microfuge for 5 min at 4°C .
Resuspend the DNAs with approximately 60 µL of 1X TE buffer .
Store the DNAs at 4°C .
Add 57 µL of proteinase K and 29 µL of 10 % Na sarcosyl and mix .
Dry the pellets briefly ( 10 - 15 min ) in the vacuum desiccator to remove the EtOH .
Save the supernatants .
Discard the supernatants .
Incubate at room temperature for 60 min .
Incubate the samples at 60 - 65°C for 60 min .
Mix by inversion .
Centrifuge for 5 min at 4°C in the microfuge .
Mix by inversion .
Place the last extraction into 2.0 mL microfuge tubes .
Mix well and hold at - 20°C overnight .
Discard the supernatants .
If the DNA doesn’t go into solution overnight , centrifuge in the microfuge for 15 min at 4°C .
Remove the supernatants to clean tubes .
Discard the pellets .
