DNA Prep

Chlamydomonas DNA Isolation

For TLN100 Rotor (1/2″ x 1 1/2″ Quick-seal tubes)
and TLA100.3 Rotor (1/2″ x 1 1/4″ Quick Seal tubes)

Part 1

  1. Grow cells, with aeration, in 250 ml of the appropriate growth medium, until the culture becomes dark green. Stationary phase cells work, but DNA yield from older cultures tends to decrease.
  2. Harvest cells in sterile, 250 ml round bottomed bottles. Spin in Sorval with IEC 949 rotor at 2.8 krpm for 12 minutes.
  3. Resuspend the cells in 10 ml of ddH20 and transfer each suspension to a sterile, disposable, 15 ml conical tube. Pellet the cells in the clinical centrifuge at full speed for 5 minutes. Decant the supernatant and adjust the volume of the cells to 2 ml (TLN100) or 1.7 ml (TLA100.3) with ddH20.
  4. Resuspend the cells by vortexing each tube with a 6″ Pasteur pipette inside the tube. Leave the pipette in the tube for the following step.
  5. Lyse the cells by adding 350 µl (TLN100) or 312 µl (TLA100.3) of 20% SDS. Vortex the tube briefly (2 seconds) and immediately add 500 µl (TLN100) or 454 µl (TLA100.3) of 5X Extraction Buffer. Vortex again to ensure complete mixing before lysing the next tube of cells.
     5X Extraction Buffer
    Reagent [stock] 100 ml [final]
    Tris 1 M 34.5 ml 0.345 M
    NaCl 5 M 27.6 ml 1.38 M
    EDTA 0.5 M 27.6 ml 0.138 M
    ddH20 — 10.3 ml —
  6. Mix gently for 30 – 60 minutes at 42° in the warm air shaker. Note: DNA yield decreases if incubation exceeds 60 minutes.
  7. Add 3.1g (TLN100) or 2.66g (TLA100.3) CsCl to each tube. Mix gently, for 10 minutes, at 42° C to dissolve the CsCl2.
  8. Add 150 µl of 10 mg/ml Ethidium bromide to each Quick-seal tube using a syringe. Use 3 ml syringes and 18 gauge needles to transfer the cell lysates to the Quick seal tubes. If a tube is not full, top it off with ddH20, leaving a small air space at the top.
  9. Balance pairs of tubes (Difference <= 0.1 g) and seal. Spin in the ultracentrifuge at 23°C.
     TLN100  90,000 rpm for 4 hours
     OR
     75,000 rpm for 8 hours
     TLA100.3  75,000 rpm for 8 hours

Part 2

  1. Visualize the DNA band using a long-wave UV light source. Note that the DNA is the pink, NOT the white band. Regular room lights may be sufficient to see the band. Pull the band using a 1 ml syringe and and 18 ga needle. Put the DNA in 1.5 ml Epindorf tube. Try to keep the total volume in a tube under 400 µl.
  2. Extract the Ethidium bromide using NaCl-saturated isopropanol. Repeat until the top (isopropanol) layer is no longer pink.
  3. Estimate the volume in each tube (X µl). Add X µl of ddH20 to each tube. If solution is cloudy extract once with phenol/chloroform.
  4. Add X µl isopropanol to each tube. Mix gently, by inverting.
  5. Let the tubes stand at room temperature for at least 15 minutes.
  6. Spin in a microfuge for 10 minutes.
  7. Decant the supernatants and wash the pellets with 70% EtOH.
  8. Dry the pellets in the SpeedVac.
  9. Resuspend each pellet in 200 µl of TE. Allow the pellets to go into solution for at least 2 days at 4°C. To help the DNA dissolve, tap the tubes occasionally. To avoid shearing, do not vortex the samples. Before quantitating the [DNA], remove insoluble material by centrifugation.

This page is, as always, Copyright (c) 1996 by Craig D. Amundsen. All rights reserved.