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Reagents and Solutions

use deionized or double-distilled water in all recipes

Tris-MgCl2 buffer (Pfosser et al. 1995)

0.2 M Tris 4.84 g
4 mM MgCl2 . 6H2O 162.64 mg
0.5% Triton X-100 1 ml

  • adjust volume to 200 ml 
  • adjust pH to 7.5 
  • filter through a 0.22 µm filter; store at 4°C

Lysis buffer LB01 (Dolezel et al., 1989)

15 mM Tris 363.4 mg
2 mM Na2EDTA 148.9 mg
0.5 mM spermine tetrahydrochloride 34.8 mg
80 mM KCl 1.193 g
20 mM NaCl 233.8 mg
0.1% (v/v) Triton X-100 200 µl

  • adjust volume to 200 ml 
  • adjust to pH 7.5 with 1N HCl 
  • add 220 µl ß-mercaptoethanol (15 mM) 
  • filter through a 0.22 µm filter 
  • store at -20°C in 10 ml aliquots

Otto Buffer I

0.1M citric acid monohydrate 4.2 g
0.5% (v/v) Tween 20 1 ml

  • adjust volume to 200 ml 
  • filter through a 0.22 µm filter; store at 4°C

Otto Buffer II

0.4M Na2HPO4 .12H2O 28.65 g

  • adjust volume to 200 ml 
  • filter through a 0.22 µm filter 
  • store at room temperature 
  • Note: A DNA fluorochrome can be added prior to final volume adjustment. In that case, the solution should be stored in darkness.

Tris buffer

100 mM NaCl 1.461 g
10 mM Na2EDTA 930.6 mg
0.1% (v/v) Triton X-100 250 µl
10 mM Tris 302.85 mg

  • adjust volume to 500 ml 
  • adjust pH to 7.5 
  • store at 4°C

Formaldehyde fixative (4% v/v)

Dilute stock formaldehyde (MERCK cat. no. 1.04003.) in Tris buffer to a final concentration of 4% (v/v)

  • The formaldehyde fixative should be freshly prepared.

CRBC buffer I

140 mM NaCl 1.637 g
10 mM sodium citrate 588.2 mg
1 mM Tris 24.23 mg

  • adjust volume to 200 ml 
  • adjust pH to 7.1

CRBC buffer II

140 mM NaCl 0.41 g
5% (v/v) Triton X-100 2.5 ml

  • adjust volume to 50 ml

CRBC buffer III

320 mM sucrose 54.77 g
15 mM MgSO4 . 7 H2O 1.85 g
15 mM ß-mercaptoethanol 530 µl
1 mM Tris 587.3 mg

  • adjust volume to 500 ml 
  • adjust pH to 7.1

DAPI stock solution

0.1 mg/ml DAPI 5 mg

  • disolve in 50 ml H2O 
  • filter through a 0.22 µm filter to remove small particles 
  • store at -20°C in 1 ml aliquots 
  • DAPI is available from Molecular Probes, Inc., (Eugene, Oregon).

Propidoum iodide stock solution

1 mg/ml propidium iodide 50 mg

  • disolve in 50 ml H2O 
  • filter through a 0.22 µm filter to remove small particles 
  • store at -20°C in 0.5 ml aliquots 
  • PI is available from Molecular Probes, Inc., (Eugene, Oregon).

RNase stock solution

1 mg/ml RNase (IIA Sigma) 25 mg

  • disolve in 25 ml H2O 
  • filter through a 0.22 µm filter to remove small particles 
  • heat to 90°C for 15 min to inactivate DNases 
  • store at -20°C in 0.5 ml aliquots