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The diluted compounds were mixed by repeated aspiration and dispensation using a 384-well P30 dispensing head on the Evolution-P3 (EP3) liquid handling platform (Perkin Elmer), and then 15 µl of each compound were transferred to the wells of assay plates.
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To achieve the 92-µM concentration, 23 nL of compound was transferred twice from the highest concentration compound plate into each well of the assay plates.
Using a Platemate Plus (Thermofisher Scientific), 500 nL of each test compound was transferred into assay plates (384 clear polystyrene plates) along with standard inhibitor and DMSO in the appropriate control wells.
A working stock (2 μL of 2 n m to 50 μ m final concentration) of each test compound was transferred to an intermediate 384-well plate using a Matrix Platemate Plus (Thermo) and pre-diluted with 38 μL minimum essential media (MEM).
Target compounds were transferred in an ion trap analyzer via an atmospheric pressure electrospray interface (AP-ESI).
Some of these compounds were transferred to females during copulation.
Some of these compounds were transferred to female cuticles in high amounts during copulation.
Compounds were transferred at 50 nl vol from 1 mmol/l Stock using a Pin tool (V&P).
Next, the pooled compounds were transferred to the reaction plates and mixed (2 μL, to give a final concentration of 1.25 μM for each molecule).
The compounds were transferred into drug plates by acoustic droplet ejection using an Echo 520 liquid handler (LABCYTE, Sunnyvale, CA, USA).
From this, 0.25 μl of the compounds were transferred to all wells using a Cartesian Hummingbird (Genomics Solution) before 10 μl of either enzyme/peptide mix or peptide alone (control) was added to the assay plates.
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