Daniel:Protocols/HiMWDNAExtract

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High MW DNA Extraction[edit]

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Protocol 3-Small Amounts of Cells[edit]

  • According to protocol it is for suspension or trypsinized cells (1000-10,000,000)
  1. Pellet Cells
    1. Trypsinize Cells with 0.5mL TrypLE; Incubate 3 min at 37C
    2. Passage cells if desired
    3. From the rest, put remainder of cells in 15 mL tube and spin down for 3 min at 1000xg
    4. Resuspend cells in 200 uL 1X PBS
  2. Before Starting
    1. Chill 100% EtOH and 80% EtOH in -20C freezer
    2. Thaw RNase (if stored at -20C) and keep on ice
    3. Equilibrate heat blocks or water baths; one to 37C and one to 65C
  3. Isolation of DNA
    1. Add 350 uL solution A to cell suspension and vortex in 1 sec intervals until evenly disbursed
    2. Incubate at 65C for 10 minutes
    3. Add 150uL Solution B and vortex vigorously until the precipitate moves freely in the tube, and the sample is uniformly viscous (10 sec-1 min)
    4. Add 500 uL chloroform and vortex until viscosity decreases and the mixture is homogeneous (10 sec-1 min)
    5. Centrifuge at max speed for 10-20 minutes at 4C to separate phases. Transfer the upper phase into a fresh microcentrifuge tube
  4. DNA Precipitation
    1. To the DNA solution, add 1 mL of 100% EtOH (-20C) and vortex briefly
    2. Incubate on ice for 30 minutes
    3. Centrifuge at max speed for 10-15 minutes at 4C. Remove the ethonal from the pellet with a drawn-out Pasteur pipette
    4. Add 500 uL of 80% EtOH (-20C) and mix by inverting the tube 3-5 times
    5. Centrifuge at max speed for 3-5 minutes. Save the pellet and remove the 80% ethanol with drawn out Pasteur pipette
    6. Centrifuge at max speed for 3-5 minutes at 4C. Remove residual ethanol with a pipettor. Let air dry for 5 minutes.
    7. Resuspend the pellet in 100 uL TE buffer. Add 2 uL of a 2 mg/mL RNase to bring concentration to 40 ug/mL
    8. Incubate at 37C for 30 minutes.
  5. Store at 4 C