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.
