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Kansas State University

C-BANDING PROTOCOL

The following protocol describes the standard C-banding protocol routinely used in our laboratory that gives consistant results for most plant species.

  1. Germinate seeds in petri dishes on moist filter paper for 3-4 days.
  2. Collect root tips when 1.5-2.5-cm long and pretreat with 0.05% colchicine for 3 h at room temperature. The colchicine pretreatment results in a lower mitotic index compared with ice water pretreatment but give better chromosome morphology and band contrast.
  3. Fix in Carnoy's I for at least 1 h, but complete metaphase spreads are more easily obtained when the material is stored in fixative solution for a longer time (2 weeks to several months) at 4°C. For meiotic chromosomes, fix anthers that are at the appropriate stage of division without pretreatment in Carnoy's I.
  4. Make squash preparations in 45% acetic acid; a 2-3-min pretreatment with 45% acetic acid before squashing softens the tissue and makes squashing easier. If possible, remove the very tip of the root with a razor blade, squeeze the meristematic tissue out with a scalpel, and squash using only gentle heat. Check quality of preparations under phase contrast.
  5. Remove cover slips by placing on dry ice until frozen.
  6. Immediately place slides in 99% ethanol. Slides should be kept in ethanol overnight, and the staining procedure continued the next day.
  7. Air dry the slides for several minutes.
  8. Incubate slides for 1 min in 0.2 N HCl in a water bath at 60°C. The time and temperature of the treatment is critical for good contrast. Prepare a 2 N HCl stock solution, which is 86 mL concentrated HCl per 500 mL distilled water. This solution can be stored for several months and should be diluted 1:9 before use to obtain a 0.2 N treatment solution.
  9. Wash briefly in distilled water.
  10. Incubate the slides for 7 min in a saturated Ba(OH)2 at room temperature. Prepare a saturated Ba(OH)2 solution in distilled water, stir for 30 min at room temperature, and filter before use. This solution needs to be prepared fresh each time.
  11. Wash briefly in distilled water.
  12. Incubate slides for 30 min in 2 X SSC in a water bath at 60°C.
    13. 20 X SSC stock solution
    tri-sodium-citrate-2-hydrate
    Na3C6H5O7·2H2O
    88.2 g
    sodium chloride
    NaCl
    175.3 g
    distilled water
    dH2O
    to 1 L
  13. Move slides directly from 2 X SSC into the staining solution, which is a 10% solution of Giemsa stain (BDH, Giemsa's stain improved R66 solution 'Gurr', stock #35086 4X (from Gallard-Schlesinger Industries, Inc; Carle Place, NY 800-645-0344) in Soerensen phosphate buffer, pH 7.2. Begin with a low Giemsa concentration and control the staining under the microscope. If necessary, add more Giemsa stain. Staining time will vary (10-45 min) between different cells on the same slide and also between different slides. Slides should be individually checked every few minutes for best contrast. A staining time of about 30 min is optimal.
    14. Soerensen's buffer
        Stock Stain
    Part A sodium phosphate dibasic (Na2HOP4) 9.47 g / L H2O   58 mL
    Part B potassium dihydrogen phosphate (KH2PO4) 9.07 g / L H2O
    42 mL
  14. Dip the slides in distilled water to rinse and air dry.
  15. For permanent slides, soak in xylene and mount in Permount. Most slides can be stored for several years without loss of contrast. However, destaing may occur, so analyzing and recording banding patterns immedially after mounting is recommended.
  16. Microphotographs should be taken with a high-resolution film such as Kodak Imagelink HQ microfilm 1461 or Kodak Technical Pan film 2415.