9/6/2020 0 Comments Serial Dilutions Chemistry
Mixing in the center allows the dispensed liquid to be more evenly distributed in the sample, thus increasing the likelihood of efficient mixing.
Serial Dilutions Chemistry Serial Dilution OfIC50 assays, commonly used to evaluate drug efficacy, and assay development procedures, as well as standard-curve generation, involve the serial dilution of compounds, proteins, or detection agents.These processes can be streamlined by utilizing automated liquid-handling equipment with serial dilution capabilities. With each sequential serial dilution step, transfer inaccuracies lead to less accurate and less precise dispensing. The result is that the highest dilutions will have the most inaccurate results. To compensate for this error possibility, longer mixing times are required, which then increases the time required to perform the serial dilution. These challenges greatly limit the throughput capacity of an automated serial dilution system. With the platforms VWorks software, the application allowed the total control of liquid transfer and mixing heights and speeds, which allowed efficient exploration of mixing parameters. ![]() The starting volume was 300 L, and 200 L tips were utilized for the transfer (150 L, a 1:2 dilution) and mixing steps (190 L). There are two main components of an accurate and precise serial dilution: the accuracy and precision of the transfer and the efficiency of mixing. Transfers were previously determined to have a precision and accuracy of 99 at this volume; any observed deviations in precision and accuracy were due to error propagation from ineffective mixing. The Coefficient of Variance (CV) of each column indicated the precision of the mixing step. The CV also provided information on the propagation of error across a platethe CV increased sharply across the plate if mixing was incomplete. A calibration curve was prepared, and each experimental dilution concentration was plotted against the standard curve to determine the real concentrations in each column. The first experiment varied the number of mixing cycles between 3 and 20. The average precision (averaging CVs for columns 110) improved asymptotically as the number of mix cycles increased. Three mixes before each transfer yielded an average CV of 11.8, while 20 mixes gave a considerably better CV of 1.7. The accuracy ratio is an average of the concentration of the diluted column compared to the previous columna perfect serial dilution has an accuracy ratio of 1:2.00 across the entire plate. The accuracy ratio of the plate improved with more mix cycles, improving from 1:1.85 to 1:2.01. The 20-mix cycle protocol required 20 minutes per plate, while a three-mix cycle protocol required less than six minutes. Efforts were then focused on the factors that could improve the three-mix cycle protocol to produce accuracy and precision results consistent with the 20-mix cycle protocol. ![]() At a height of 3 mm from the bottom of the well, the average precision was 3.9. The precision worsened as the tip distance from the bottom of the well decreased, reaching a CV of 15 at a height of 0.1 mm. Accuracy tracked with precision, and the higher mix height also improved the accuracy ratio to 1.95. This trend is possible because the higher dispense height ensures that more of the sample was circulated by the mix cycle. If the mix occurs close to the bottom of the plate, the dispensed liquid is pulled back into the tip during the aspiration.
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