suitable for the extraction of urea and ammonium after foliar urea application
The completed extraction procedure yields 2.0 ml extract for each sample.
If ammonium is to be determined in the extracts, gloves should be worn at all times to avoid ammonium contaminations.
Sampling + sample preparation
Remove several leaves from the treated plants.
Wash leaves briefly in a small water container to remove surface urea. Dry leaves with paper towels.
Cut leaves into pieces, randomise and freeze in a plastic bag - use part or all of the material for subsequent steps.
Grinding and weighing
Grind frozen leaf material in liquid nitrogen.
Weigh out approximately 0.1 g of ground material (in a frozen state) in 2.0 ml microfuge tubes. (NOTE: The weighing of frozen leaf material is a bit tricky, cool down the spatula used in liquid nitrogen and work quickly. Slight melting is usually not a problem - urease activity (at least in potato leaves) is completely abolished when the material thaws). Record the exact weight.
Add 1 ml of water to the material and freeze tubes in liquid nitrogen. At this point the extraction can easily be interrupted to be continued at a later time. Extracts can be stored at -70°C.
Extractions
A tube cap holder is applied over the cap of the frozen tubes to prevent them from opening in the subsequent boiling step.
The frozen samples are placed in a boiling water bath for 3 min, tubes may be inverted 2-3 times.
The tubes are immediately placed on ice and the cap holders are removed.
Each sample is vortexed for 30 seconds and then centrifuged for 1.5 minutes (15000 g). The supernatant is removed into a new 2 ml Eppendorf tube on ice.
0.5 ml water is added to the pellet, which is re-extracted by vortexing for 30 seconds. Again the samples are centrifuged, the supernatant is removed and pooled with the first supernatant stored on ice.
The pellet is again re-extracted with 0.5 ml water as in the previous step.
