Standard Operating Procedure: Harvesting of Arabidopsis Thaliana Plants

Standard Operating Procedure GARNet 5

Analysing Arabidopsis thaliana plants for Carotenoids.

Author: Jennie Lewis

Last Modified: January 2001

Standard Operating Procedure:

Analysing Arabidopsis thaliana plants for Carotenoids.

Materials: Equipment:

1.5ml Eppendorf tubesGilson pipettes and tips

MethanolBalance

Tris-HCl (50mM pH7.5 adjusted with 1MHCl)Whirlimix

ChloroformCentrifuge

Polystyrene BoxSpeedie Vac

IceHPLC with Diode Array Detector

ChloroformColumn: ODS-5 5μ 250x4.6mm

Glass pasteur pipettes Guard Cartridge: ODS-5 5μ

Glass screw top vials

Injection vials and caps

Tapered low volume inserts

Acetonitrile

Polished Water

Triethylamine

Ethyl Acetate

Procedure:

Step 1: Plant Extraction

1.1Label three 1.5ml eppendorf tubes with the plant material to be analysed and Replicate 1, 2 or 3.

1.2Fill a polystyrene box with ice and leave to stand on bench.

1.3Weigh out 10mg of freeze dried material, prepared for analysis following SOP GARNet 4, into each labelled eppendorf tube.

1.4To each tube add 200μl Tris-HCl (50mM pH 7.5 adjusted with IM HCl).

1.5Mix the plant material and solvent by holding the tubes in a bench top whirlimix for 1 minute.

1.6Place the tubes in the ice box for 10 minutes.

1.7To each tube add 800μl Chloroform.

1.8Repeat steps 1.5 and 1.6.

1.9Centrifuge the tubes at 3000g for 5 minutes.

1.10Label glass screw top vials with plant material and replicate number.

1.11With a glass pasteur pipette transfer the coloured chloroform fraction, from the bottom of the eppendorf tube, to the appropriately labelled glass vial.

1.12Add 800μl Chloroform to each eppendorf tube containing plant material and aqueous phase.

1.13To re-extract the plant material repeat steps 1.5, 1.6 and 1.9.

1.14To pool the chloroform fractions repeat step 1.11.

1.15Place the vials in a speedie vac and run the machine until all the liquid has evaporated, leaving a residue at the bottom of the vial.

1.16Store the glass vials, containing the dry residues, at -80°C until analysis can be carried out.

1.17Prior to analysis add 200μl Ethyl Acetate to the dry residues, mix the vials for 1 minute using a bench top whirlimix.

1.18Label injection vials with the appropriate plant material and replicate number and into each place a low volume insert.

1.19Transfer 50μl of the sample to the appropriately labelled injection vial and cap the vial.

Step 2: HPLC Anlaysis

2.1Make up Mobile Phases A: 9:1 Acetonitrile:Water (containing 1% triethylamine) [Store in a brown 1L Duran bottle] and B: Ethyl Acetate.

2.2Set up the HPLC with a ODS-5 250x4.6mm column with in line guard cartdrige.

2.3Programme the HPLC to run from 0-100% Mobile Phase B over 15 minutes at a flow rate of 1ml/min.

2.4Set the diode array detector to scan at 250-550nm and to selectively monitor at 425 and 450nm.

2.5Equilibrate the column in 100% Mobile Phase A until a flat base line is maintained.

2.6Inject 25μl of each prepared sample, from the injection vial, in to the HPLC system.

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