Method for efficiently extracting internal mucus layers of arabidopsis thaliana
A technology of Arabidopsis thaliana and mucus, which is applied in the field of efficient extraction of the inner mucus layer of Arabidopsis thaliana, which can solve the problems such as the difficulty of extracting the inner mucus layer, and achieve the effects of simplifying labor, shortening extraction time, and reducing costs
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Embodiment 1
[0054] Example 1: Research on the extraction efficiency of Arabidopsis mucus layer by different extraction methods
[0055] (1) Preparation of extraction reagents: Prepare the following extraction reagents with purified water: 0.05 MEDTA, 0.2% ammonium oxalate (w / v), 0.01 M NaOH, 0.05 M HCl.
[0056] (2) Extraction of Arabidopsis mucus layer with different chemical extraction reagents: Accurately weigh 5mg of Arabidopsis seeds, add 1mL of pure water, shake on a shaker at 150rpm for 5min, centrifuge at 3000rpm, take the supernatant as the outer layer of mucus Floor. Wash the seeds 3 times with purified water, add 1mL of corresponding reagents: purified water, 0.05MEDTA, 0.2% ammonium oxalate (w / v), 0.01MNaOH, 0.05MHCl, shake at 200rpm on a shaker at 40°C for 1h, centrifuge at 3000rpm, take Clear, for the inner mucus layer.
[0057] (3) Extraction of Arabidopsis mucus layer by ultrasonic treatment: Accurately weigh 5 mg of Arabidopsis seeds, add 1 mL of pure water, place on a ...
Embodiment 2
[0060] Example 2: Ultrasonic extraction of mucus layer of Arabidopsis thaliana
[0061] (1) Extraction of the outer mucus layer: Accurately weigh 5 mg of Arabidopsis seeds, add 1 mL of pure water, shake on a shaker at 150 rpm for 5 minutes, centrifuge at 3000 rpm, and take the supernatant to form the outer mucus layer.
[0062] (2) The same tube of seed samples undergoes mucus layer extraction at different ultrasonic stages: for the seeds in step (1), after cumulative ultrasonication of 5S, 10S, 20S, 40S, 1min, 2min, 4min, and 6min, the supernatants were taken respectively as internal layer mucus layer, and wash the seeds with purified water twice between each ultrasonic interval.
[0063] (3) Changes in the mucus layer of the same tube of seed samples after different ultrasonic stages: for the seeds in step (1), after accumulating ultrasonic waves of 0S, 5S, 10S, 20S, 40S, 1min, 2min, 4min, and 6min, the seeds were taken respectively, and each Wash the seeds with purified wa...
Embodiment 3
[0066] Example 3: Fluorescence Observation of Arabidopsis Seed Mucus Layer
[0067] (1) After Arabidopsis seeds were shaken with water for 5 minutes to remove the outer mucus layer, they were washed twice with 0.1M phosphate buffer solution (Phosphate Buffer Solution, PBS; 1L: 0.218gKH2PO4, 1.463gK2HPO4, 29.22gNaCl, pH7.2). 5min;
[0068] (2) Soak the seeds with fresh 5% (w / v) skimmed milk, shake on a slow shaker for 1 hour; remove the skimmed milk, and wash the seeds with PBS buffer for 5 minutes;
[0069] (3) Primary antibody incubation: incubate the seeds with 20-fold diluted mouse anti-RGI antibody CCRC-M14 (ComplexCarbohydrate Research Center) for 1 h, and wash the seeds with PBS buffer 5 times to wash away the unbound primary antibody;
[0070] (4) Secondary antibody incubation: incubate with 50-fold diluted FITC-goat anti-mouse antibody (Comfort Century, Cat. No. CW0113S) for 1 h, and wash the seeds with PBS buffer 5 times to wash away the unbound secondary antibody; ...
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