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When the homogenization was completed, the extract and a fragrance was added to the solution at 55°C, and the solution was again homogenized for 5 min using a homo mixer at 4,000 4,500 rpm.
A pH conditioning agent was added at 45 50°C, and the solution was again homogenized for 3 min using a homo mixer at 3,500 4,000 rpm.
Samples were again homogenized, using the TeSeE™ Precess 48 homogenizing system [26].
The homogenate was centrifuged at 4000 g for 20 min. The supernatant was saved, and the pellet was again homogenized and centrifuged.
The supernatant was again homogenized with two strokes of a Teflon pistle, centrifuged at 1000 g for 10 min, and the resulting supernatant was centrifuged for 10 min at 8000 g.
The supernatant was discarded and the pellet resuspended in 300 μl buffer A and left on ice for 10 min before the pellet was again homogenized and centrifuged at 3000 rpm for 30 min at 4 °C.
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After 5 days of exposure to P. aeruginosa, stool was again collected, homogenized in 1 mL 1% protease peptone, serially diluted in 1% proteose peptone and dilutions plated on TSA and LB agar (with 0.015 mg gentamicin/mL (Research Product International, Mt. Prospect, IL) for PA14 transposon insertion mutants) to measure levels of GI colonization by P. aeruginosa.
The pellet was resuspended in 5 mL of buffer A, homogenized again in a Dounce homogenizer, and transferred to 1.5 mL of buffer AS to collect the nuclei by centrifugation.
The tissue extracts were centrifuge at 1,500 g for 5 min and the pellets were homogenized again in 1 ml buffer for 8 strokes and centrifuged again.
It was homogenized again when the solution was poured and the film was cast in Pyrex dish.
The gel was prepared from 3cm3 acrylamide reagent (Sigma-Aldrich) mixed with 8cm3 Tris-borate-EDTA (TBE) Buffer 0.5× (Promega Corporation, Madison, USA) into erlenmayer, homogenised then temed (Sigma-Aldrich) 20 μl and homogenized again.
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