##BLOCKS= 4 Note: Quantitation of double-stranded DNA using Quant-iT PicoGreen Reagent Invitrogen (Molecular Probes) MATERIALS o Quant-iT PicoGreen dsDNA Assay Kit, including lambda DNA standard (Invitrogen cat. #P7589 or P11496) o Black 96-well plate (Greiner Bio-One, cat. # 655096) o Brown or amber (light-blocking) microcentrifuge tubes METHODS Set up the protocol: o Select Wells to Read and Assay Plate Type by clicking on "Settings" and locating the options on the left side of the screen. o Click the Template button to open a window where you can assign wells of the microplate to pre-set template groups using the drop-down menu to select the appropriate template group. There are preconfigured template groups in the PicoGreen Fluorescence protocol including Standards, Unknowns, and Unknowns_NoDiln (for undiluted samples). Assigning wells to pre-set template groups populates group tables in the protocol with the corresponding data acquired when the microplate is read. Prepare the assay The method for this assay follows the instructions in the product information sheet for Quant-iT PicoGreen dsDNA Reagent and Kits from Molecular Probes, except that the assay volume is proportionately reduced from 2.0 mL to 200 uL to fit a 96-well microplate format. o Prepare 1X TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) by diluting the o concentrated buffer from the kit 20-fold with distilled DNase-free water, as required by Molecular Probes. o Prepare an aqueous working solution of Quant-iT PicoGreen reagent by making a 200-fold dilution of the concentrated DMSO solution in TE buffer (prepared above). Preparation of the solution in a plastic container, rather than glass, is recommended, as the reagent may adsorb to glass surfaces. Protect the solution from light by using amber or brown tubes, or by covering with foil. This solution should be used within a few hours of its preparation. o DNA standard curve: Prepare a 2 µg/mL stock solution of dsDNA in TE. The lambda DNA standard provided with the kit can be diluted 50-fold in TE to make the 2 µg/mL solution. Note: in some cases it may be preferable to make the standard curve using DNA similar to the type being assayed. o A high-range standard curve may be prepared from 1 ng/mL to 1 µg/mL, or a low-range standard curve may be prepared from 25 pg/mL to 25 ng/mL. For the high-range curve, follow the dilution scheme shown in the PicoGreen product insert; for the low-range curve, dilute the 2 µg/mL solution 40-fold to yield a 50 ng/mL solution, and refer to the alternative dilution scheme in the product insert. o Pipet standards into a solid black 96-well microplate at 100 µL per well, preferably in triplicate. Be sure to include a set of buffer blank wells containing TE only (no DNA). o Add 100 µL of the aqueous working solution of Quant-iT PicoGreen reagent to each well. Mix well by trituration or plate shaker and incubate for 2 to 5 minutes at room temperature, protected from light. Read the microplate " Make sure the purple plate adapter is in the microplate reader drawer. Place the microplate in the drawer. " Click the Read button in the SoftMax Pro software. The instrument will read the plate and the relative fluorescence units will be displayed in the Plate section of the protocol. Analyze the data o After the microplate has been read, the relative fluorescence units (RFUs) will be displayed in the Plate section. The data will be analyzed in the Group Tables that were created when the template was set up. o Standards assigned in the Template (and thus displayed in the Standards group table) will be automatically plotted in the Standard Curve section of the protocol. A linear curve fit is applied by default, but a log-log fit may be used when plotting a standard curve over a wide dynamic range. Curve fits are chosen from the drop-down Curve Fit menu in the graph section's tool bar. ----------------------------------------- READER SUITABILITY: All SpectraMax readers with fluorescence capability. PROTOCOL REVISION HISTORY: v 1.1; Imported from SMP 5.4.2 April 2011 (CLO & ELM) v 1.2; Emission wavelength changed from 540 nm to 525 nm. (CLO) ~End Plate: Plate01 1.3 PlateFormat Endpoint Fluorescence FALSE Reduced FALSE 1 1 535 1 12 96 485 Manual 0 1 8 1 2 3 4 5 6 7 8 9 10 11 12 960874 870214 322628 3834736 1001297 1645009 2564803 276866 19815620 -13428 -13447 -13420 30624 334107 2699408 6532039 6104545 2241062 908118 9508843 9976684 -13436 -13367 -13391 342542 7961883 6016136 10518554 8157747 1504190 1753246 5379288 4160517 -13354 -13401 -13301 7887012 6823398 6451660 6545008 8550473 179453 1001350 3393857 1362236 -13341 -13301 -13436 33414 6058776 5338043 1405758 4154540 197595 1046013 286432 411461 -13256 -13296 -13357 3889640 607730 9766018 8712689 833890 1176562 1628909 3694279 165277 -13405 -13386 -13394 2419210 2058096 4068652 6086470 5809783 2228421 96531 5213651 82663 -13362 -13325 -13405 9791161 1253346 6431202 6438697 4132413 499941 820610 -13273 0 -13347 -13210 -13386 ~End Group: Standards Sample Concentration BackCalcConc Wells RFU_Values MeanRFUValue SD CV 01 50.000 49.860 A9 19815620.000 19815620.000 0.000 0.0 02 25.000 25.937 B9 9976684.000 9976684.000 0.000 0.0 03 12.500 11.794 C9 4160517.000 4160517.000 0.000 0.0 04 6.250 4.990 D9 1362236.000 1362236.000 0.000 0.0 05 3.125 2.678 E9 411461.000 411461.000 0.000 0.0 06 1.563 2.080 F9 165277.000 165277.000 0.000 0.0 07 0.781 1.879 G9 82663.000 82663.000 0.000 0.0 Group Summaries ~End Group: Unknowns_NoDiln Sample Wells RFU_Values Concentration MeanConc SD CV 01 A1 960874.000 4.014 4.014 0.000 0.0 02 B1 30624.000 1.752 1.752 0.000 0.0 03 C1 342542.000 2.511 2.511 0.000 0.0 04 D1 7887012.000 20.855 20.855 0.000 0.0 05 E1 33414.000 1.759 1.759 0.000 0.0 06 F1 3889640.000 11.136 11.136 0.000 0.0 07 G1 2419210.000 7.560 7.560 0.000 0.0 08 H1 9791161.000 25.485 25.485 0.000 0.0 09 A2 870214.000 3.794 3.794 0.000 0.0 10 B2 334107.000 2.490 2.490 0.000 0.0 11 C2 7961883.000 21.038 21.038 0.000 0.0 12 D2 6823398.000 18.269 18.269 0.000 0.0 13 E2 6058776.000 16.410 16.410 0.000 0.0 14 F2 607730.000 3.156 3.156 0.000 0.0 15 G2 2058096.000 6.682 6.682 0.000 0.0 16 H2 1253346.000 4.726 4.726 0.000 0.0 17 A3 322628.000 2.462 2.462 0.000 0.0 18 B3 2699408.000 8.242 8.242 0.000 0.0 19 C3 6016136.000 16.306 16.306 0.000 0.0 20 D3 6451660.000 17.365 17.365 0.000 0.0 21 E3 5338043.000 14.658 14.658 0.000 0.0 22 F3 9766018.000 25.424 25.424 0.000 0.0 23 G3 4068652.000 11.571 11.571 0.000 0.0 24 H3 6431202.000 17.316 17.316 0.000 0.0 25 A4 3834736.000 11.002 11.002 0.000 0.0 26 B4 6532039.000 17.561 17.561 0.000 0.0 27 C4 10518554.000 27.254 27.254 0.000 0.0 28 D4 6545008.000 17.592 17.592 0.000 0.0 29 E4 1405758.000 5.096 5.096 0.000 0.0 30 F4 8712689.000 22.863 22.863 0.000 0.0 31 G4 6086470.000 16.477 16.477 0.000 0.0 32 H4 6438697.000 17.334 17.334 0.000 0.0 33 A5 1001297.000 4.113 4.113 0.000 0.0 34 B5 6104545.000 16.521 16.521 0.000 0.0 35 C5 8157747.000 21.514 21.514 0.000 0.0 36 D5 8550473.000 22.469 22.469 0.000 0.0 37 E5 4154540.000 11.780 11.780 0.000 0.0 38 F5 833890.000 3.706 3.706 0.000 0.0 39 G5 5809783.000 15.805 15.805 0.000 0.0 40 H5 4132413.000 11.726 11.726 0.000 0.0 41 A6 1645009.000 5.678 5.678 0.000 0.0 42 B6 2241062.000 7.127 7.127 0.000 0.0 43 C6 1504190.000 5.335 5.335 0.000 0.0 44 D6 179453.000 2.114 2.114 0.000 0.0 45 E6 197595.000 2.158 2.158 0.000 0.0 46 F6 1176562.000 4.539 4.539 0.000 0.0 47 G6 2228421.000 7.096 7.096 0.000 0.0 48 H6 499941.000 2.894 2.894 0.000 0.0 49 A7 2564803.000 7.914 7.914 0.000 0.0 50 B7 908118.000 3.886 3.886 0.000 0.0 51 C7 1753246.000 5.941 5.941 0.000 0.0 52 D7 1001350.000 4.113 4.113 0.000 0.0 53 E7 1046013.000 4.221 4.221 0.000 0.0 54 F7 1628909.000 5.639 5.639 0.000 0.0 55 G7 96531.000 1.913 1.913 0.000 0.0 56 H7 820610.000 3.673 3.673 0.000 0.0 57 A8 276866.000 2.351 2.351 0.000 0.0 58 B8 9508843.000 24.799 24.799 0.000 0.0 59 C8 5379288.000 14.758 14.758 0.000 0.0 60 D8 3393857.000 9.930 9.930 0.000 0.0 61 E8 286432.000 2.374 2.374 0.000 0.0 62 F8 3694279.000 10.661 10.661 0.000 0.0 63 G8 5213651.000 14.355 14.355 0.000 0.0 64 H8 -13273.000 1.646 1.646 0.000 0.0 Group Summaries ~End Original Filename: HiSeq241_plate3_library_conc_11-24-2015; Date Last Saved: 11/24/2015 1:12:38 PM