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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

MODIFIED N-BANDING TECHNIQUE FOR SEQUENTIAL CHROMOSOME BANDING AND IN SITU HYBRIDIZATION (ISH)

  1. Remove cover slips from the slides that have been stored in a -70°C freezer with a razor blade; and dry the slides in an ethanol series (75%, 95%, and 100% ethanol, 5 min each).
  2. Incubate slides in hot phosphate buffer (1M NaH2PO4) at 92 ± 2°C for 2 min, rinse in tap water, and immediately stain with Giemsa solution.
  3. Briefly rinse slides with tap water and dry with a hand puff.
  4. Record the N-banding patterns of well-spread cells with a high-resolution objective that does not require a cover slip (i.e., Olympus NC Plan Apo 60 objective).
  5. Destain slides in an ethanol series (75%, 95%, and 100% ethanol, 5 min each; slides also can be briefly dipped several times in 45% acetic acid before the ethanol series to remove the Giemsa stain).