The following protocol describes the standard N-banding protocol routinely used in our laboratory for chromosome identification in wheat and wheat relatives and will give satisfactory results even in the hands of an inexperiencesd worker.
- Germinate seeds in petri dishes on moist filter paper for 3-4 days.
- Collect root tips when 1.5-2.5-cm long and pretreat for 24 h in an ice-water bath.
- Fix in Carnoy's I at room temperature for 1-3 days and then freeze or refrigerate until use.
- Stain root tips in 1% acetocarmine for 1-2 h at room temperature, or overnight in at 4°C.
- Make squash preparations in 45% acetic acid.
- Remove cover slips by either the dry ice method, dipping slides into liquid nitrogen for about 10 sec, or placing slides into a freezer (-70°C) for 3 min.
- Incubate slides in hot 45% acetic acid for 3 min in a water bath at 50°C.
- Transfer slides to 95% ethanol at room temperature for 10 min.
- Air dry slides overnight.
- Incubate slides in phosphate buffer (120 g NaH2PO4 / L distilled water) for 2 min at 94°C in a water bath.
- Rinse in tap water and air dry.
- Stain with Leishman's stain (BDH #R03087) in Soerensen's phosphate buffer for about 20 min. The staining solution is diluted 5 X with water and Leishman's stain added to a final concentration of 15%).
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
- Rinse briefly in tap water and air dry.
- For recording of N-bands, a cover slip can be mounted using a drop of xylene or preparations can be observed without a cover slip using the appropriate lenses.