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After weighing, tissue samples were frozen, lyophilized and pulverized.
After weighing, tissue samples (~50 mg) were placed in a liquid nitrogen pre-cooled 14 mL round-bottom culture tube, followed by addition of 1 mL TRIzol Reagent (Invitrogen, Carlsbad, CA, USA).
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The within day variation of weighing tissues in the 10 50 mg range, using a Mettler AC100 precision weighing device (Mettler Instruments, Tiel, the Netherlands), was estimated to be 0.2-0.6 0.2-0.6
The weighed tissue debridement sample was dissected into small pieces and placed in a 1.5 ml micro-centrifuge tube.
In the procedure of heat treatment in Distilled water (H1 method), the weighed tissue debridement sample was thoroughly minced and suspended in 100 µl of sterile distilled water.
The weighed tissue debridement specimen was suspended in 1 ml of sterile TN150 buffer and 0.3 g of sterile zirconium beads (diameter, 0.1 mm) were added.
The weighed tissue debridement sample was suspended in 600 µl of sterile TNES buffer [10 mM Tris HCl pH = 7.5, 400 mM NaCl, 100 mM EDTA, 0.5% SDS] and 20 µl of proteinase K and mixed by inverting.
Radioactivity concentrations were measured in weighed tissue samples, whole blood, and plasma using a Packard Cobra II automated gamma counter, with correction to radioactivity at the time of tracer injection relative to the entire injected dose (%ID/g).
Furthermore, the image-derived results were confirmed by well counting on weighed tissue samples (based on tissues samples placed in RNAlater) from each rat.
DNA was extracted from weighed tissue samples using DNeasy Blood and Tissue (Qiagen) following the manufacturer's recommendations.
Briefly, weighed tissue pieces were lyophilized, digested in nitric acid overnight, and heated at 90°C for 20 minutes.
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