Phaseolus polytene chromosomes

Pretreatments

RNase pretreatment

Preparation

  • thaw the RNase solution
  • pre-heat the humid chamber to 37C

Procedure

  • apply 30 l of RNase-solution onto the slide and cover with cover-slip
  • incubate preparations in humid chamber for 45 min at 37C and pre-warm 70 ml PBS in water bath at 60C
  • rinse preparations for 30 min in PBS at 60C
  • rinse slides 2 times for 5 min in PBS at room temperature

Pepsin/HCl pretreatment

Preparation

  • prepare fresh 70 mL solution of Pepsin/HCl
  • pre-warm water bath at 37C /li>

Procedure

  • rinse slides 2x in 0.1 N HCl at room temperature
  • incubate preparations for 3 h in staining jar with Pepsin/HCl solution at 37C
  • rinse slides 2x in 0.1 N HCl and 1x in BT buffer at room temperature
  • dehydrate preparations by immersion in ethanol-series (5 min each step) and air-dry

Material

  • glass staining jars, water-bath at 37C and 70C
  • 200l pipette with autoclaved tips
  • humid chamber

Chemicals and solutions

  • ethanol-series: 70%, 85% und 99% methylated ethanol
  • 0.1 N HCl
  • PBS: 0.13 NaCl, 0.007 M NaH2PO4, 0.003 M Na2HPO4, pH 7.4
  • Pepsin porcine, 17 milliAnson U/mg, 2x cryst. lyophil (SERVA 31820)
  • Pepsin-solution: 1 mg Pespin/mL in 0.1 N HCl
  • RNase-solution: 100 g/ml, DNase-free in Aqua dest.
09/22/2018 23:32  Phaseolus polytene chromosomes by Mario Nenno. © 2008- All rights reserved.
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