Phaseolus polytene chromosomes

Fluorochrome banding

For fluorchrome banding with 4',6-Diamidin-2-phenyl-indol (DAPI) polytene chromosomes have been incubated for 10 min.

In order to get a differential fluorochrome banding of blue, pink and white preparations have been stained in a mixture of DAPI and propidiumiodid (DAPI/PI).
As material were used chromosome prerpations at least 24 hours after pretreatment or slides used previously for FISH.

Procedure

  • prepare new staining solution and store protected form light
  • incubate chromosome preparations in 60 ml staining solution in a dark place at least overnight at room temperature
  • rinse slides in PBS and remove access of liquid
  • cover chromosome preparations in 10 µl of mounting medium and leave in the dark for at least 30 min before examination in fluorescence microscope under UV

Chemicals and solutions

  • DABCO: Diazabicyclo[2.2.2]octan (Sigma)
  • DAPI stock solution: 0.2 mg/ ml
  • 4',6-Diamidin-2-phenyl-indol2HClH20 (Serva) in Aqua dest.
  • Propidiumiodid stock solution: 1 mg/ml Propidiumiodid in Aqua dest.
  • staining solution: 20 µl DAPI stock solution and 20 µl PI stock solution in 60 ml PBS
  • PBS: 0.13 M NaCl, 0.007 M NaH2PO4, 0,03 M Na2HPO4, pH 7.4
  • Mounting medium, either: a) Vectashield (Serva) or b) 1 part Tris-HCl pH 7.4 and 9 parts Glycerin with 2.3% (w/v) DABCO and 0.02% NaN3

12/18/2018 19:10  Phaseolus polytene chromosomes by Mario Nenno. © 2008- All rights reserved.
Texts and images can be used for free for personal and educational use if the work on which is based this website
is cited properly as given under References. Any commercial use must be requested beforehand.