拟南芥角质测定方法.docVIP

Analysis of Arabidopsis cutin Note: Need 1-6g of Arabidopsis tissues (stems and/or leaves) ~ 100 mg dry residue per g of leaves can be obtained (starting from about 3 g of leaves) IMPORTANT: ALWAYS RINSE ALL YOUR GLASSWARE AND CAPS WITH CHLOROFORM BEFORE USING. Tissue delipidation: Days 1-3 1. Preheat water bath at 80-85C. 2. Weigh leaves (~0.5g). 3. Put into pre-weighted 25ml tubes, add isopropanol (25ml per g tissue, ~12 ml) and 80C for 10-15 min. 4. Let tubes cool down and grind tissue with the Polytron. Do not grind too thin. Add a small stirring bar to each tube. 5. Stir for 1-2h. 7. Centrifuge 10 min at 2000 rpm. 8. Add same volume of isopropanol and stir overnight 9. Centrifuge 10 min at 2000 rpm. 10. Add CHCl3/CH3OH (2/1 v/v) to residue (25ml per g of tissue, ~12 ml) and stir the whole day (RT). 11. Centrifuge 10 min at 2000 rpm 12. Add CHCl3/CH3OH (1/2 v/v) to residue (25ml per g of tissue, ~12 ml) and stir overnight at RT. 13. Centrifuge 10 min at 2000 rpm, remove solvent and let it dry under hood overnight. 14. Dry finally in a vacuum desiccator for two days (can keep several weeks). Depolymerization: Methanolysis with sodium methoxide. 1. Weight the tubes containing the dry residue to know the amount of dry residue (important for further calculations). 3. Add standards: 25 ul C17:0 ME (1mg/ml stock), and 25 ul pentadecanolactone (1 mg/ml stock) 3. Add 0.9 ml methyl acetate, 1.5 ml sodium methoxide and 3.6 ml methanol to each tube. Why do we add MeOAc? Any water in the system will produce NaOH from NaOMe.: NaOMe + H2O ? NaOH + MeOH This NaOH will be rapidly removed by saponification of methyl acetate to produce sodium acetate and methanol. ROH + MeOAc ? ROAc + MeOH 4. Heat 2 h at 60 °C. 5. Let samples cool down. 6. Add methylene dichloride (8-10 ml) and glacial acetic acid to pH 4-5 to extract the fatty acid methyl esters. Fill the rest of the tube with saline solution (0.5M NaCl). 7. Vortex and centrifuge 10 min at 2000 rpm. 8. Trasfer the