ISOLATION OF PLANT GENOMIC DNA FOR GENOMIC IN
SITU HYBRIDIZATION (GISH)
- 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.
- 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
- 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.
- 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).
- Put sample into a centrifuge tube and spin at 10,000 rpm for 15
min at room temperature.
- 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.
- 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.
- Add 500 µL phenol, mix well, and spin for 5 min; transfer
the supernatant to another tube.
- Add 250 µL each of phenol and chloroform, mix well, and spin
for 5 min; transfer the supernatant to another tube.
- Add 500 µL chloroform, mix well, and spin for 5 min; transfer
the supernatant to a 10-mL tube.
- 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.
- 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.
- Determine the DNA concentration by using spectrophotometer or by
electrophoresis on a minigel.