Viral DNA Miniprep Procedure
Infect 60 mL of chlorella with 200 µL of viral single plaque isolates.
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.
Save the supernatants.
Save the unused portion of the lysates.
Add 10% NP-40 (or Triton X-100) to the lysate supernatants to a final concentration of 1%.
Centrifuge the material in Beckman Ti50.2 rotors at 15,000 rpm (~27,000 rcfmax), 75 min, 4°C.
Discard the supernatants.
Resuspend the virus pellets with 1.0 mL of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2.
Transfer 350 µL of the resuspended virus to 1.5 mL screw-cap microfuge tubes and adjust the final volume to 500 µL with 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2.
Add 8.8 µL of DNAse I and mix.
Incubate at room temperature for 60 min.
Add 6.0 µL of 500 mM EDTA, pH 8.0 to the samples and mix.
Add 56.6 µL of proteinase K and 29.0 µL of 10% Na sarcosyl and mix.
Incubate the samples at 60-65°C for 60 min.
Add 300 µL of buffer-saturated phenol and 300 µL of CHCl3:Isoamyl alcohol (24:1) to the tubes.
Mix by inversion.
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.
Mix by inversion and centrifuge for 5 min at 4°C in the microfuge.
Remove the upper aqueous layers to clean tubes and repeat the CHCl3:Isoamyl alcohol extraction 1X.
Place the last extraction into 2.0 mL microfuge tubes.
Add 66 µL of 3 M NaOAc to each tube.
Precipitate the DNAs with 2X volumes (approximately 1350 µL) of 100% EtOH.
Mix well and hold at -20°C overnight.
Centrifuge the tubes in the microfuge for 10-15 min at 4°C to pellet the DNAs.
Discard the supernatants.
Wash the DNA pellets 1X with 1000 µL of 70% EtOH in the microfuge for 5 min at 4°C.
Dry the pellets briefly (10-15 min) in the vacuum desiccator or the speed vac (5 min) to remove the EtOH.
Resuspend the DNAs with approximately 60 µL of 1X TE buffer.
If the DNA doesn’t go into solution overnight, centrifuge in the microfuge for 15 min at 4°C and remove the supernatants to clean tubes.
Discard the pellets.
Store the DNAs at 4°C.
