Standard Operating Procedure GARNet 6
Analysis of Arabidopsis thaliana for Phenylpropanoids.
Author: Jennie Lewis
Last Modified: February 2002
Standard Operating Procedure:
Analysing Arabidopsis thaliana plants for Phenylpropanoids.
Materials:
Equipment:
1.5ml eppendorf tubes
5ml glass test tubes
Glass pasteur pipettes
Methanol
Polished Water
Acetic Acid
Ethyl Acetate
Acetonitrile
3,5 Dihydroxybenzoic Acid
Injection vials and caps
Tapered low volume inserts
Gilson pipettes and tips
Centrifuge
Balance
Whirlimix
Speedie Vac
HPLC with Diode Array Detector
Column: Lichrosorb RP C18 10μ 250x4.6mm
Guard Cartridge: Lichrosorb RP C18 10μ
HPLC with Diode Array Detector
Procedure:
Step 1: Plant Extraction
1.1
Label three 1.5ml eppendorf tubes with the plant material to be
analysed and Replicate 1, 2 or 3.
1.2
Make up a 2mg/ml solution of 3,5 Dihydroxybenzoic acid in 50:50
Acetonitrile:Water (with 2.5% acetic acid).
1.3
Weigh out 500mg fresh, frozen plant material or 50mg freeze dried
plant material, prepared for analysis following SOP GARnet 4, into
each labelled eppendorf tube.
1.4
To each tube add 900μl 70:30 Methanol:Water (containing 2.5% acetic
acid).
1.5
To each tube add 100μl of Internal Standard 3,5-Dihydroxybenzoic acid
(concentration 2mg/ml in 50:50 ACN:Water with 2.5% Acteic Acid).
1.6
Mix the plant material and solvent by holding the tubes in a bench top
whirlimix for 1 minute.
1.7
Leave the tubes to stand for 10 minutes on the bench.
1.8
Repeat step 1.6.
1.9
Centifuge the tubes at 18000g for 3 minutes.
1.10
Label 5ml glass test tubes with the plant material and replicate number.
Page 1 of 3
Standard Operating Procedure GARNet 6
Analysis of Arabidopsis thaliana for Phenylpropanoids.
Author: Jennie Lewis
Last Modified: February 2002
1.11
Transfer 500μl of supernantant to the correctly labelled tube.
1.12
Add to the supernatant 1ml distilled water (containing 2.5% acetic acid)
and 1ml ethyl acetate.
1.13
Mix the tubes for 1 minute using a bench top whirlimix.
1.14
Leave the tubes on the bench until clear partitioning can be seen.
1.15
Label a second set of 5ml test tubes with the appropriate plant material
and replicate number.
1.16
Remove the ethyl acetate layer with a glass pasteur pipette and
transfer to the correctly labelled clean tube. Use a clean pipette for
each replicate.
1.17
Place the tubes in a speedie vac and run the machine until all the liquid
has evaporated, leaving a green residue in the bottom of the tube. This
usually take 30 – 60 minutes.
1.18
Reconstitute the samples in 50μl 50:50 ACN:Water (with 2.5% acteic
acid).
1.19
Label injection vials with the appropriate plant material and replicate
number and into each place a tapered low volume glass insert.
1.20
Transfer the reconstituted samples, with a glass pasteur pipette, to the
appropriately labelled injection vial and cap the vial.
Step 2: HPLC Analysis
2.1
Make up Mobile Phases A: 2.5% Acetic Acid and B: Acetonitrile.
2.2
Set up the HPLC with a Lichrosorb RP C18, 10μ, 250x4.6mm column
with in line guard cartridge.
2.3
Programme the HPLC with the following gradient and flow rate of
1ml/min.
Page 2 of 3
Standard Operating Procedure GARNet 6
Analysis of Arabidopsis thaliana for Phenylpropanoids.
Author: Jennie Lewis
Last Modified: February 2002
Time (minutes)
0
5
7
47
52
54
57
60
% Mobile Phase A
100
83
82
67
0
0
100
100
% Mobile Phase B
0
17
18
33
100
100
0
0
2.4
Set the diode array detector to scan at 250-550nm and to selectively
monitor at 332nm.
2.5
Equilibrate the column in 100% Mobile Phase A until a flat base line is
maintained.
2.6
Inject 25μl of each prepared sample in to the HPLC system.
Page 3 of 3