Agilent Well to well reproducibility using a white 96 well microplate Manual
Contents
1. 808 099 Sample A10 836 170 Sample C10 827 059 Sample E10 833 147 Sample G10 809 085 Sample A11 840 729 Sample C11 847 120 Sample E11 813 528 Sample G11 830 481 Sample A12 842 289 Sample C12 843 965 Sample E12 825 361 Sample G12 824 091 Sample B12 833 176 Sample D12 833 152 Sample F12 836 795 Sample H12 825 334 Sample B11 829 479 Sample D11 832 562 Sample F11 830 509 Sample H11 832 613 Sample B10 833 872 Sample D10 817 077 Sample F10 819 648 Sample H10 845 312 Sample B9 830 945 Sample D9 810 861 Sample F9 812 878 Sample H9 794 645 Sample B8 819 317 Sample D8 810 779 Sample F8 807 364 Sample H8 808 268 Sample B7 823 151 Sample D7 807 006 Sample F7 804 247 Sample H7 811 349 Sample B6 815 508 Sample D6 816 661 Sample F6 808 477 Sample H6 809 721 Sample B5 825 550 Sample D5 790 917 Sample F5 788 024 Sample H5 789 566 Sample B4 823 444 Sample D4 820 737 Sample F4 810 156 Sample H4 815 523 Sample B3 838 167 Sample D3 813 145 Sample F3 807 742 Sample H3 810 123 Sample B2 834 430 Sample D2 812 319 Sample F2 808 832 Sample H2 815 381 Sample B1 847 012 Sample D1 801 833 Sample F1 813 325 Sample H1 821 572 Average 820 571 Standard 13 724 RSD 1 67 deviation www agilent com chem Agilent Technologies Inc 2001 2011 Published March 2011 Publication Number SI A 1825 Agilent Technologies2. Author Jeff Comerford Agilent Technologies Mulgrave 3170 Australia Well to well repeatability using a white 96 well microplate Application Note Introduction In order to obtain results of the highest precision well to well repeatability is extremely important Where conventional instruments have repeatability in the order of 5 the Agilent Cary Eclipse Microplate reader has repeatability for every single well in a 96 well plate of less than 2 Parameters that may affect repeatability are 1 Variation in well to well volume 2 Variation in meniscus shape from well to well 3 Alignment of the microplate Parameters 1 and 2 can be minimized by adopting correct laboratory procedures whereas parameter 3 is a function of the microplate reader alignment The Agilent Cary Eclipse addresses the alignment issue via a sophisticated yet easy to use software procedure which aligns a plate of specific type accurately within the accessory This procedure accounts for any plate variations due to manufacture Microplate variability may cause deviations from the general assumption that the wells in any 3 corners of a standard plate form a right angle see Figure 1 Deviations from this right angle assumption can result in clipping of the sides of the well which affects the well to well repeatability ais Agilent Technologies ABCDEFGH 8 3 6 b 4 3 2 1 S POOO 0000000 Figure 1 Schematic of a 96 well microplate showing tha
3. e performed once if the total well volume remains the same between each assay and if repeatability of less than 2 is required Otherwise well to well repeatability in the order of 4 is obtained without this optimization The Cary Eclipse Help has videos which describe how to align your plate reader for optimum results Summary The Cary Eclipse Microplate reader by virtue of its all reflective optical design and superior software Auto alignment procedure is one of the most precise microplate readers available Well to well repeatability for every single well in a White 96 well microplate can be achieved to within less than 2 Appendix Table 2 Results from a white 96 well plate using 1m M fluoroscein in each well Sample name Intensity Sample name Intensity Sample name Intensity Sample name Intensity Sample A1 834 312 Sample C1 841 139 Sample E1 808 301 Sample G1 809 856 Sample A2 833 922 Sample C2 836 107 Sample E2 810 293 Sample G2 818 193 Sample A3 840 697 Sample C3 835 781 Sample E3 825 734 Sample G3 817 207 Sample A4 832 314 Sample C4 834 735 Sample E4 815 352 Sample G4 816 786 Sample A5 822 183 Sample C5 819 614 Sample E5 800 961 Sample G5 183 971 Sample A6 821 491 Sample C6 818 373 Sample E6 807 017 Sample G6 805 200 Sample A7 832 983 Sample C7 828 628 Sample E7 822 020 Sample G7 810 040 Sample A8 824 343 Sample C8 835 146 Sample E8 807 907 Sample G8 816 446 Sample A9 830 013 Sample C9 830 064 Sample E9 818 018 Sample G9
4. t Well A1 A12 and H12 form a right angle Method and Results A volume of 300 uL of a 1 UM solution of Fluorescein was pippetted into each well of a Greiner white 96 well microplate The plate was aligned using the Auto align procedure in the Cary Eclipse Align application and the intensity was measured for each well using the parameters shown in Table 1 The results obtained from a typical run are shown in Table 2 see Appendix Table 1 Instrument parameters used in repeatability experiment Instrument Parameter Setting Excitation Wavelength 490 nm Emission Wavelength 520 nm Excitation Slit 10 nm Emission Slit 10 nm Averaging time 0 1 s Excitation Filter Auto Emission Filter Auto PMT Voltage 400V Discussion The experiment was repeated a total of 6 times and similar results were obtained The average RSD from the six runs was calculated to be 1 75 The best results are obtained by ensuring that the microplate reader is correctly aligned for both the plate type being used and the volume of solution in each well NOTE The volume of solution should be the same from well to well and air bubbles should be avoided Use a multi tip pipette for reproducible volume delivery Aligning the microplate reader for the plate type is a simple Auto align software procedure Optimizing the well volume involves adjusting the emission horizontal and vertical mirror adjustments on the accessory However this procedure only needs to b
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Agilent Well to well reproducibility using a white 96 well microplate Manual