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Kansas State University

ISOLATION OF PLANT GENOMIC DNA FOR GENOMIC IN SITU HYBRIDIZATION (GISH)

  1. Grind 5 g of plant leaf tissue in liquid nitrogen (do not let the plant material thaw) and pour powder into a 250 mL flask.
  2. Suspend powdered material in 15 mL of 2 X CTAB buffer and incubate in a water bath at 65°C for 1 h under slow shaking.
    • 2 X CTAB buffer is 1.4 M NaCl, 100 mM Tris pH 8.0, 2% CTAB (hexadecyltrimethylamonium bromide), 20 mM EDTA, 0.5% Na bisulfide, 1% 2-mercaptoethanol (2-me).
    • When making the CTAB buffer, it is recommended to add the NaCl after the other ingredients except 2-me are in solution. Do not add the 2-me until just before use.
    • The solution without 2-me can be stored at room temperature.
  3. Let the flasks cool down to room temperature, add 15-mL chloroform/isoamyl alcohol (24:1) and swirl the flask until an emulsion is formed (place the flask on a rotary shaker for 15 min at room temperature).
  4. Put sample into a centrifuge tube and spin at 10,000 rpm for 15 min at room temperature.
  5. Transfer the supernatant to a clean tube and add 2/3 volumes of isopropyl alcohol; invert the tubes several times; hook out the precipitated DNA; and transfer to a clean 10-mL tube.
  6. Wash the DNA pellet twice with 70% ethanol. Pour off the ethanol and let the pellet air dry for 1 h.
    add 500 µL of TE and transfer the DNA into a 1.5-mL tube; add 5 µL RNase A (10 mg/mL) and leave at 4°C overnight or at 37°C for 1 h.
  7. Add 500 µL phenol, mix well, and spin for 5 min; transfer the supernatant to another tube.
  8. Add 250 µL each of phenol and chloroform, mix well, and spin for 5 min; transfer the supernatant to another tube.
  9. Add 500 µL chloroform, mix well, and spin for 5 min; transfer the supernatant to a 10-mL tube.
  10. Add TE to a final volume of 3 mL, then add 1/10 volume of 3 M NaAc and 2 volumes of 100% ethanol. Invert the tube several times.
  11. Wash the precipitated DNA twice with 70% ethanol; transfer the dDNA to a 1.5-mL tube; dry the DNA pellet; and dissolve into 500 ml TE.
    • As a rule, the cleaner the genomic DNA, the better the biotin-labeled probe. Therefore, the isolated DNA may be further purified by using a CsCl-purification procedure. However, the quality of DNA isolated according to the protocol described above is adequate for GISH in our laboratory.
  12. Determine the DNA concentration by using spectrophotometer or by electrophoresis on a minigel.