protocol_103.txt

Isolation Of Total DNA From NC64A Chlorella
Inoculate 500 mL flasks with NC64A chlorella, each flask to contain 360 mL of cells at 1.2 X 106 cells/mL in MBBM.
Incubate the flasks at 25°C for 72 hours, with continuous light and shaking.
Count the cells.
Concentrate aliquots of NC64A chlorella, each aliquot to contain 6.0 X 109 cells.
If the cells are to be infected with virus, infection should be at an moi (multiplicity of infection) of 3-5.
Centrifuge the samples in the Sorvall GSA rotor at 5,000 rpm, 5 min, 4°C to harvest the cells.
Wash the cells 1X with sterile d-H2O in the Sorvall HB-4 rotor at 5,000 rpm, 5 min, 4°C.
Quick freeze the cells pellets with liquid N2 and store the frozen pellets at -80°C until ready for processing.
Resuspend each frozen sample with 5.0 mL of 50 mM NaHPO4, pH 7.4, 2.0 M NaCl.
Heat the samples at 65°C for 30 min (leave in the MSK flasks during the heating).
Break the samples a second time in the MSK for 30 sec with CO2 cooling.
Recover the homogenates to clean tubes (SS34 plastic tubes).
Remove a 0.3 mL aliquot from each sample to microfuge tubes for determination of the 			original DNA concentration for each sample (use the fluorometric procedure).
Treat each sample with 500 µL of proteinase K for 60 min at 37°C (add 200 µL/sample).
Heat the samples at 65°C for 5 min.
Add 20% SDS to each sample to a final concentration of 1% (add 500 µL/sample).
Add 2.7 mL of 5 M KOAc to each sample, mix well (a final concentration of 1 M).
Incubate the samples in the cold room for 30 min.
Treat the samples with RNAse at 200 µg/mL for 2 hours at 37°C (add 200 µL/sample).
Precipitate the DNAs in the samples by adding 2X volumes of 100% EtOH (approximately 30 mL/sample).
Centrifuge the tubes in the Sorvall HB-4 rotor at 10,000 rpm, 15 min, 4°C.
Resuspend each DNA sample with 3.5 mL of 50 mM Tris-HCl, pH 8.0, 10 mM EDTA.
Dialyze the samples overnight at 4°C against several changes of 50 mM Tris-HCl, pH 8.0, 10 mM EDTA.
Add 375 µL of 3 M NaOAc to each sample.
Centrifuge the samples in the Sorvall SS34 rotor at 10,000 rpm, 20 min, 4°C.
Wash the DNA pellets 1X with 10 mL of 70% EtOH in the Sorvall SS34 rotor at 10,000 rpm, 5 min, 4°C.
Dry the pellets briefly (10-15 min) in the vacuum desiccator to remove the EtOH.
Determine the DNA concentration of the samples using the fluorometric procedure.
Run CsCl gradients.
Centrifuge the required volume of cells for each aliquot in the Sorvall GSA rotor at 5,000 rpm, 5 min, 4°C.
Resuspend each aliquot with 160 mL of fresh MBBM.
Incubate the flasks for 45-60 min at 25°C for the cells to acclimate to the new media.
Incubate the flasks for the desired length of time at 25°C with continuous light and shaking.
Discard the supernatants.
Break the cells in the MSK mechanical homogenizer with 5.0 gm of 0.3 mm glass beads for 60 sec, with CO2 cooling.
Wash the glass beads with 50 mM NaHPO4, pH 7.4, 2.0 M NaCl.
Add the washes to the homogenates.
Centrifuge the sample in the Sorvall SS34 rotor at 14,000 rpm, 20 min, 4°C.
Decant the supernatants to clean tubes.
Store at -20°C for 2-3 hours.
Discard the supernatants.
Precipitate the DNAs by adding an equal volume of isopropanol to each tube.
Mix well and hold at room temperature for 30 min.
Discard the supernatants.
Resuspend the pellets with 2.0 mL of 1X TE buffer.