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 .
