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Wheat Genetics Resource Center

Kansas State University
Department of Plant Pathology

4024 Throckmorton
1712 Claflin Road
Manhattan, KS 66506-5502
785-532-6176
785-532-5692 fax
wgrc@k-state.edu

Wheat Genetics Resource Center
Kansas Wheat Innovation Center

1990 Kimball Ave
Manhattan, KS 66502
785-320-4383

ISOLATION OF PLASMID (PROBE) DNA FOR IN SITU HYBRIDIZATION (ISH)

  1. Inoculate a 2-mL culture in a Kaput tube from a single colony and place it on a 37°C shaker overnight.
  2. Inoculate a 400-mL culture in a 1 L flask by pouring the 2-mL preculture into the flask; allow 4 to 5 hrs incubation at 37°C while shaking.
  3. Harvest the cells by centrifugating for 10 min at 6,000 rpm at 4°C. Decant the supernatant and place at an angle on ice to allow the remaining liquid to drain away from the pellet. Remove the remaining liquid with a sterile pipette after several minutes.
  4. Resuspend the pellet in 5 mL of solution I (50 mM glucose, 25 mM Tris pH 8.0, 10 mM EDTA); and transfer the culture into a 50-mL Nalgene polyallomer centrifuge tube.
  5. Add 10 mL of solution II (O.2 N NaOH, 1 % SDS), cover tube with Parafilm, and gently invert repeatedly to mix; allow the solution to set on ice for 10 min.
  6. Add 7.5 mL of solution III (3 M NaAc pH 4.8), cover tube with parafilm, and mix by inverting gently.
  7. Centrifuge tube at 12,000 rpm for 15 min at 4°C, pour the supernatant into a second 50-mL tube, add 11-mL isopropanol, cover with parafilm and mix by inverting gently centrifuge tube for 5 min at 5,000 rpm at 4°C, decant supernatant, rinse the pellet in 70% ethanol; allow the tube to drain for 10 min, lypholize to near dryness; disslove the pellet in 4.5-mL TE.
  8. Add 4.7 g of CcCl and 0.4 mL of a 10 mg/ml ethidium bromide solution in TE (the density of the sample is between 1.55 and 1.59 g/mL, add TE or CsCl to adjust; plasmid DNA without CsCl purification also can be used for biotin-labeling, however better results are obtained when CsCl purified plasmid DNA is used).
  9. Tranfer the solution into a Beckman '13 x51' polyallomer quick-seal tube, fill to just below the neck (the tube now should weigh 9.45-9.6 g), and seal.
  10. Centrifuge tube at 20°C at 65,000 rpm for 4 hrs or 55,000 rpm overnight.
  11. Remove the plasmid band from tube with a needle and syringe, and transfer to a 1.5-mL tube.
  12. Add 1 volume of saturated isopropanol (mix equal volumes of isopropanol and 20 X SSC pH 7.0) and mix well. Centrifuge, or allow to stand for several minutes. Remove upper phase with pipette.
  13. Repeat the extraction 4 to5 times, or until the pink color in the propanol disappears.
  14. Dilute the DNA sample with 2 volumes of TE, add 1/10 volume of 3M NaAc pH 7 and mix well.
  15. Add 2 volumes of 95% ethanol and mix well.
  16. Centrifuge for 5 min, drain tube, rinse the pellet and sides of the tube with 70% ethanol, drain the ethanol, and dry the pellet.
  17. Dissolve the DNA into 400 µL TE, add 20 µL RNase A (10 mg/mL), mix, and incubate for 15 min at room temperature.
  18. Add an equal volume of a 1:1 phenol:chloroform solution, mix well, centrifuge briefly, and transfer the upper phase to another tube.
  19. Add an equal volume of chloroform, mix well, centrifuge briefly, and transfer the upper phase to another tube.
  20. Precipitate the DNA with 3 M NaAc and ethanol and dissolve the pellet in 400 µL TE.
  21. Determine the DNA concentration by using spectrophotometer or by electrophoresis on a minigel.