This procedure is generalized. Use of little experiments is possible. Please contact us at lipid@ksu.edu (please allow in a subject line) to inquire.
Take up to 8 leaves (or up to 3 whole little plants- see NOTE #2); speedily immerse in 3 ml 75ºC (preheated) isopropanol with 0.01% BHT (butylated hydroxytoluene, e.g., Sigma B1378) for 15 min. Use a 50 ml (25 x 150 mm) glass tube with a Teflon-lined cognize cap. NOTE #1: It is extremely important that the plants be ask outed directly after sampling and that the isopropanol be preheated. Plants have rattling active phospholipase D, which is activated upon wounding; failure to place the sampled weave quickly into hot isopropanol will result in generation of phosphatidic acid. NOTE #2: To do the analysis, only a fraction of an Arabidopsis leaf is needed. Use of more weave reduces variability among samples. Dry weights of 5 to 30 mg, measured as in step 7, are recommended.
come 1.5 ml anaesthetise and 0.6 ml water, twist; then agitate (shaking incubator) at room temperature for 1 hour. Transfer (long, glass Pasteur pipettes) lipid extracts to glass tubes with Teflon-lined screw-caps.
total 4 ml chloroform/methanol (2:1) with 0.01% BHT; shake 30 min.
Repeat this extraction procedure on all samples until the leaves of both sample become white, but typically apiece sample is extracted the same number of times. (Use one pipette in each sample for all extractions, leaving them in the removed extract while extracting the remaining materials.) Its OK to leave for slenderly longer than 30 min on later extractions. commonly you will need about 5 extractions, including the one with the isopropanol.
Add 1 ml 1 M KCl to the combined extract, vortex or shake, centrifuge, discard upper phase.
Add 2 ml water, vortex or shake, centrifuge, discard upper phase.
Fill tubes with nitrogen, break in in freezer; and transport to the KLRC Analytical Laboratory on dry ice....If you want to get a full essay, position it on our website: Orderessay
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