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LABORATORY PROCEDURES FOR PLANT CELL LINES

REFERENCE NO.: PC/1998/3/1.2


 TITLE: STANDARD EXTRACTION PROCEDURE FOR CALLUS AND SUSPENSION CULTURES


INTRODUCTION

For cell characterisation and authentication as well as for regular quality control of plant cell lines the cells are extracted. The extracts may be further analysed by UV spectroscopy, reverse phase HPLC or gaschromatography

PROCEDURE

1. 2 g of fresh cell material or 500 mg lyophilised cell material are weighed into a 100 ml round bottomed flask.

1.1. If non-lyophilised fresh cells are extracted the cell dry weight has to be measured by the standard procedure (PC/1998/3/1.2 Appendix 1).

2. 10 ml solvent is added to the round bottomed flask.

3. The flask is fixed in a reflux extraction apparatus.

3.1. The flask is placed into the heating mantle of the reflux extraction apparatus.

3.2. A condenser is fixed onto the flask.

3.3. An upper closing device is fixed to the condenser with an outlet into the laboratory hood to avoid the evaporation of solvent into the laboratory.

4. The cell material is extracted by reflux extraction for one hour.

4.1. Condensers are cooled by opening the water passage.

4.2 Heaters are switched on (step 1).

4.3 Reflux extraction is performed for one hour in the boiling solvent.

4.4. After one hour of extraction heaters are switched off.

4.5. When the solvent has cooled down to ambient temperature the water passage is closed.

5. The contents of the flask (solvent and cells) is filtered through filter paper wetted with the extraction solvent into a 50 ml Erlenmeyer flask.

6. Evaporated solvent is replaced by adjusting the volume to the initial value of 10 ml.

7. The extracts are filled into screw cap glass tubes (air and solvent tight) and stored.


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