Lyophilization

1. Harvest leaves from greenhouse or field grown plants. It is preferable to use young leaves without necrotic
areas or lesions, although older leaves which are not senescent may be used.
2. If the midrib is thick and tough, remove it. Cut or fold leaves into 10-15 cm sections and place in a fiberglass
screen mesh bag along with the tag identifying the sample (Aluminum foil or paper bags may be substituted if
holes are punched to allow good air flow). Place bags in an ice chest or other container with ice to keep
samples cool (but do not allow to freeze).
3. Place leaf samples in a Styrofoam container or some type of container able to hold liquid nitrogen. Add liquid
nitrogen to quick-freeze samples. Once frozen, do not allow samples to thaw until freeze-dried!
NOTE:

Leaf samples may be stored at -80•C until ready to be lyophilized.
4. Transfer frozen leaf samples to lyophilizer. Make sure that the lyophilizer is down to temperature (the
chamber is • -60•C) and pulling a good vacuum (• 10 microns Hg) before loading samples. Do not overload
lyophilizer ó make sure vacuum is always • 100 microns and condenser temperature is • -60•C. Samples
should be dry in 72 hours. Typically, fresh weight • 10X dry weight.
5. Dried leaf samples may be stored in sealed plastic bags at room temperature for a few days or, preferably, at –
20•C for several years.
6. Fill out a harvesting record sheet.
Grinding
1. Grind to a fine powder with a mechanical mill (Tecator Cyclotec Sample Mill, Model 1093), into a plastic
scintillation vial or any other appropriate plastic container that can be closed air tight.
NOTE:

If the plant material weighed less than 4 g fresh weight, grind to a powder with a mortar and pestle
in the presence of a pinch of acid washed sand.
The finer the grind, the greater the yield of DNA from a given amount of material.
2. Store ground samples tightly capped at -20•C. Samples are stable for several years.

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