DNA Extraction Procedure for Ceratopteris - Modified QIAGEN Genomic-tip Procedure
Leslie G. Hickok 4/96

The following procedure results in yields of about 25 ug per g fresh weight of 2 week old gametophytic tissue.

  1. Grind 2-3 g gametophytes in liquid nitrogen with a mortar and pestle. Harvest tissue from ca. 2 week old liquid cultures (50 mg spores 500 mL culture medium) by aspiration or filtration. If older cultures are used the males are dead and contribute no DNA but an abundance of carbohydrates. Rinse harvested tissue with 200 mL dH2O. Blot and weigh. Pre-chill mortar and pestle with liquid nitrogen. Grind tissue thoroughly with 3 or 4 additions of N2. Use powder funnel to transfer material to a 50 mL Corning tube. Material can be transferred as a slurry, however, let it thaw before adding buffer (next step). If material is not thawed, it will foam excessively. Use an open and not an insulated rack
  2. Add 25 mL Carlson Lysis Buffer. Use buffer prewarmed to 74 C. Mix - watch pressure in tube!
    Add 3 mg RNAase. (300 æl of 10 mg/mL stock).
    Add 38 uL B-mercaptoethanol. Vortex at full speed for 5-10 seconds.
  3. Incubate for 20 minutes at 74 C. If a shaking water bath is not available, mix frequently by gentle inversion.
  4. Transfer to a 40 mL Oak Ridge (OR) tube after cooled to room temperature.
    Add one volume of chloroform/isoamyalcohol (24:1) and vortex at full speed for 5-10 sec. Place flat on shaker for 5-10 minutes.
  5. Centrifuge for 10 min at 5000 x g, 4 C. Balance tubes first!
  6. Transfer upper aqueous phase to a fresh OR tube. Yield is dependent upon cleanly transferring as much of the upper layer as possible at this step!
  7. Add one volume of isopropanol (room temperature). Vortex to mix thoroughly and incubate at room temperature for 30 minutes
  8. Centrifuge for 20 minutes at 5000 x g, 4 C. Balance tubes first!
  9. Remove (draw or pour off) supernantant (Watch pellet!). Invert over KimWipe for 5-10 min and vacuum walls of tube.
  10. Resuspend pellet in 5 mL 1.0 M NaCl for 30 minutes at 62 C. Pellet should dissolve after about 15 minutes. Intermittent swirling will likely be needed. Success may depend on removing most of the ispropanol in the previous step.
  11. Cool to room temperature Add 1.7 mL dH2O and 3.3 mL Buffer QBT

    The remaining steps are from the QIAGEN Genomic-tip Protocol. Refer to that protocol for additional tips, etc.
  12. Equilibrate a QIAGEN Genomic-tip 100 (for quantities up to 100 ug DNA) with 4 mL Buffer QBT. Allow tip to empty by gravity flow
  13. Vortex the sample from step 11 for 10 seconds at maximum speed and apply to column. Allow it to enter the resin by gravity flow.
  14. Wash the column with 2X 7.5 mL Buffer QC
  15. Elute the genomic DNA with 5 mL Buffer QF that has prewarmed to 50 C. Elute into a 15 mL disposable Corning screw-top tube

    (Note: Refer to the full QIAGEN protocol for other options at this step.
  16. Add 0.7 volumes (3.5 mL) of room temperature isopropanol. Mix by inverting several times. Collect DNA by spooling onto a sealed glass pipette tip. You may want to use a dissecting microscope tp aid in collection. Remove droplets of isopropanol and transfer the DNA to a microtube in to 50 uL TE buffer, pH 8.0. Additional DNA can be recovered from the 15 mL Corning tube by centrifuging at full speed in a clinical centrifuge for 10 minutes at room temperature. Use a sealed glass pipette tip to transfer the precipitate to a microtube into 50 uL TE buffer, pH 8.0.

BUFFERS

Carlson Lysis

  • 100 mM Tris-Cl, pH 9.5
  • 2% CTAB
  • 1.4 M NaCl
  • 1% PEG 6000 or 8000
  • 20 mM EDTA
Start with 100 mL dH2O. Add 8 mL 0.5 M EDTA, pH 8.0; 40 mL 0.5 M Tris-Cl, pH 9.5; 10 mL 20% PEG 6000 or 8000, 16.36 g NaCl. Warm mixture to 50 C and then add 4 g CTAB. After CTAB is partially in solution, adjust volume to 200 mL with dH2O and autoclave.

QBT

  • 750 mM NaCl, pH 7.0
  • 50 mM MOPS
  • 15% ethanol
  • 0.15% Triton-X 100
Dissolve 4.383 g NaCl, 1.046 MOPS (free acid) in 80 mL dH2O. Adjust to pH 7.0 with NaOH. Add 15 mL absolute ethanol and 1.5 mL 10% Triton X-100 solution. Adjust volume to 100 mL with dH2O.

QC

  • 1.0 M NaCl, pH 7.0
  • 50 mM MOPS
  • < 15% ethanol
Dissolve 5.844 g NaCl and 1.046 g MOPS (free acid) in 80 mL dH2O. Adjust to pH 7.0 with NaOH. Add 15 mL absolute ethanol. Adjust volume to 100 mL with dH2O

QF

  • <1.25 M NaCl, pH 8.5
  • 50 mM Tris-Cl
  • 15% ethanol <
Dissolve 7.305 g NaCl and 0.6055 g Tris-base in 80 mL dH2O. Adjust to pH 8.5 with HCl. Allow to cool to room temperature before making final pH adjustment. Add 15 mL absolute ethanol. Adjust volume to 100 mL with dH2O