Prepare chromosome suspension

1. Harvest 1-cm root tips and transfer into deionized H2O.

Root tips must be harvested immediately after treatment with amiprophos-methyl (see Basic Protocol 1, step 12, or see Alternate Protocol 2, step 6) or ice water (see Alternate Protocol 1, step 7).

2. Immediately transfer root tips to 25 ml formaldehyde fixative and fix at 5°C for 30 min (field bean) or 20 min (garden pea, barley, and rye).

3. Wash roots in 25 ml Tris buffer three times for 5 min each at 5°C.

4. Excise root meristems and transfer them to a 5-ml polystyrene tube containing 1 ml LB01 lysis buffer.

5. Isolate chromosomes by homogenizing at 9500 rpm using a Polytron PT1200 for 15 sec (field bean and garden pea) or 10 sec (barley and rye).

6. Filter the suspension through 50-|m nylon mesh into a 5-ml polystyrene tube.

7. Store the suspension on ice.

Although the chromosome suspension can be stored overnight, it is recommended to analyze the chromosomes on the same day.

Alternatively, root tips may be homogenized using a razor blade. Transfer fixed root tips into 1.25 ml LB01 lysis buffer in a 6-cm glass petri dish. Chop meristem root tips individually using a sharp razor blade, avoiding dispersion or drying. Filter the suspension through a 50-^m nylon mesh into a polystyrene tube. Pass the suspension once through a 22-G needle to disperse intact metaphase chromosomes, and store the suspension on ice. This method is more laborious and inconvenient in species with small root tips. However, it results in higher yield of longer chromosomes in species with large chromosomes, such as field bean.

Examine quality of chromosomes

8. Transfer 50 |l chromosome suspension into a 0.5-ml PCR tube.

10. Place a small drop (~10 |l) of DAPI-stained suspension on a microscope slide.

11. Using a fluorescence microscope, observe the suspension under low magnification (10x to 20x). Do not cover with a coverslip.

The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be >5 x 105/ml. If the chromosomes are damaged (broken and/or appear as long extended fibers), the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, the fixation was too strong and should be shortened.

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