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Chromosome Isolation in Field Bean

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 4 % formaldehyde fixative and fix at 5°C for 30 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 7.5).
  5. Isolate chromosomes by homogenising at 15000 rpm for 15 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes 

Approximately 30 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation. 
Root tips may be homogenised also using a razor blade. This method is more laborious. However, it results in higher yield of longer chromosomes in field bean:

  1. Add 1.25 ml of LB01 lysis buffer into a glass petri dish (6-cm diameter).
  2. Transfer fixed root tips into the petri dish.
  3. Chop meristem root-tips individually using a sharp razor blade, avoid dispersion or drying.
  4. Filter the suspension through a 50-µm nylon mesh into a polystyrene tube.
  5. Syringe the suspension once through a 22-gauge needle to disperse intact metaphases.
  6. Store the suspension on ice.

References

Dolezel J, Cihalikova J, Lucretti S. A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba. L. Planta 188: 93 - 98 (1992)