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A 20 g of stem was thoroughly washed with Tween 20 and double-distilled water.
About 25 g of stem bark dry powder was extracted with 250 ml of methanol solvent using soxhlet extractor.
For each plant tissue sample, ~2 g of stem segments (four plants) were excised with a sterile razor blade, dehydrated in liquid nitrogen and stored at -80°C.
Total RNA was isolated from 1.5 g of stem that included bark and wood tissue using the RNeasy plant mini kit (QIAGEN) following the protocol supplied by the manufacturer.
Particularly, about 2 g of stem derived callus, grown in solid medium supplemented with NAA 2 mg/l were transferred into 50 ml of the same medium lacking agar, and used for establishing cell cultures.
Enzyme was extracted by grinding 3 g of stem and leaf tissue in liquid nitrogen, followed by suspension in 10 ml of extraction buffer (0.1% PBS Tween-20, 2% PVPP, 1 mM EDTA, 1 mM PMSF, 1 μg leupeptin, 100 mM ascorbic acid, pH 7.4).
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A mixture of approximately 1 g of roots, 1 g of stems and 1 g of leaves was ground to a fine powder under liquid nitrogen.
For RLM-RACE, callus tissues were used as starting materials (Jayaraman and Mohamed 2015), while in gene expression, 1 g of woody stem was used in RNA extraction.
WSC were extracted from 0.1 g of ground stem material by extracting once with 8 mL of 80% ethanol at 80 °C in water bath followed by two extractions with 8 mL distilled water at 70 °C.
A previous study was conducted with 4 g of dried stem of Aristolochia manshuriensis (which contained 4 mg of AA) administered daily for 5 days in 170 g female Wistar rats.
Figure 2 Non-structural carbohydrate content (mg / g dry weight of stem) was measured at non-submerged control (C) and at the end of 14 days (14 d) and 20 days (20 d) of submergence.
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