##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 -13638 -10196 17664411 6256225 4895780 7241120 560974 8771305 532022 6346631 7631749 5589831 -3290 -13632 9079739 8170135 815105 9654830 8033679 6367628 138358 4915560 10256394 7114824 -13833 -13810 4017519 9143976 -298 11545902 -1473 3053851 208422 9749405 10058218 2858459 -13709 -13593 1303862 5483934 285208 11838575 181376 10030732 644131 10369650 2235445 13717188 -13776 -13803 403826 8395771 3623907 13472356 1149472 10805595 123051 10286245 2104511 3670941 -13852 -13860 160453 10504625 11603604 2045303 836710 10511306 167109 11518421 12493073 -13648 -13803 -13772 73343 2728066 10999131 1102815 619814 9358223 131380 11998439 6795279 -13508 -13846 -13780 0 6899911 12336037 3493238 10453997 485 962968 10256299 1184423 -13544 ~End Group: Standards Sample Concentration BackCalcConc Wells RFU_Values MeanRFUValue SD CV 01 50.000 49.602 A3 17664411.000 17664411.000 0.000 0.0 02 25.000 26.193 B3 9079739.000 9079739.000 0.000 0.0 03 12.500 12.390 C3 4017519.000 4017519.000 0.000 0.0 04 6.250 4.990 D3 1303862.000 1303862.000 0.000 0.0 05 3.125 2.536 E3 403826.000 403826.000 0.000 0.0 06 1.563 1.872 F3 160453.000 160453.000 0.000 0.0 07 0.781 1.635 G3 73343.000 73343.000 0.000 0.0 Group Summaries ~End Group: Unknowns_NoDiln Sample Wells RFU_Values Concentration MeanConc SD CV 01 A4 6256225.000 18.494 18.494 0.000 0.0 02 A5 4895780.000 14.785 14.785 0.000 0.0 03 A6 7241120.000 21.180 21.180 0.000 0.0 04 A7 560974.000 2.965 2.965 0.000 0.0 05 A8 8771305.000 25.352 25.352 0.000 0.0 06 A9 532022.000 2.886 2.886 0.000 0.0 07 A10 6346631.000 18.741 18.741 0.000 0.0 08 A11 7631749.000 22.245 22.245 0.000 0.0 09 A12 5589831.000 16.677 16.677 0.000 0.0 10 B4 8170135.000 23.713 23.713 0.000 0.0 11 B5 815105.000 3.657 3.657 0.000 0.0 12 B6 9654830.000 27.762 27.762 0.000 0.0 13 B7 8033679.000 23.341 23.341 0.000 0.0 14 B8 6367628.000 18.798 18.798 0.000 0.0 15 B9 138358.000 1.812 1.812 0.000 0.0 16 B10 4915560.000 14.839 14.839 0.000 0.0 17 B11 10256394.000 29.402 29.402 0.000 0.0 18 B12 7114824.000 20.836 20.836 0.000 0.0 19 C4 9143976.000 26.369 26.369 0.000 0.0 20 C5 -298.000 1.434 1.434 0.000 0.0 21 C6 11545902.000 32.918 32.918 0.000 0.0 22 C7 -1473.000 1.431 1.431 0.000 0.0 23 C8 3053851.000 9.762 9.762 0.000 0.0 24 C9 208422.000 2.003 2.003 0.000 0.0 25 C10 9749405.000 28.019 28.019 0.000 0.0 26 C11 10058218.000 28.862 28.862 0.000 0.0 27 C12 2858459.000 9.229 9.229 0.000 0.0 28 D4 5483934.000 16.388 16.388 0.000 0.0 29 D5 285208.000 2.213 2.213 0.000 0.0 30 D6 11838575.000 33.716 33.716 0.000 0.0 31 D7 181376.000 1.929 1.929 0.000 0.0 32 D8 10030732.000 28.787 28.787 0.000 0.0 33 D9 644131.000 3.191 3.191 0.000 0.0 34 D10 10369650.000 29.711 29.711 0.000 0.0 35 D11 2235445.000 7.530 7.530 0.000 0.0 36 D12 13717188.000 38.839 38.839 0.000 0.0 37 E4 8395771.000 24.328 24.328 0.000 0.0 38 E5 3623907.000 11.317 11.317 0.000 0.0 39 E6 13472356.000 38.171 38.171 0.000 0.0 40 E7 1149472.000 4.569 4.569 0.000 0.0 41 E8 10805595.000 30.900 30.900 0.000 0.0 42 E9 123051.000 1.770 1.770 0.000 0.0 43 E10 10286245.000 29.483 29.483 0.000 0.0 44 E11 2104511.000 7.173 7.173 0.000 0.0 45 E12 3670941.000 11.445 11.445 0.000 0.0 46 F4 10504625.000 30.079 30.079 0.000 0.0 47 F5 11603604.000 33.076 33.076 0.000 0.0 48 F6 2045303.000 7.012 7.012 0.000 0.0 49 F7 836710.000 3.716 3.716 0.000 0.0 50 F8 10511306.000 30.097 30.097 0.000 0.0 51 F9 167109.000 1.891 1.891 0.000 0.0 52 F10 11518421.000 32.843 32.843 0.000 0.0 53 F11 12493073.000 35.501 35.501 0.000 0.0 54 F12 -13648.000 1.398 1.398 0.000 0.0 55 G4 2728066.000 8.874 8.874 0.000 0.0 56 G5 10999131.000 31.427 31.427 0.000 0.0 57 G6 1102815.000 4.442 4.442 0.000 0.0 58 G7 619814.000 3.125 3.125 0.000 0.0 59 G8 9358223.000 26.953 26.953 0.000 0.0 60 G9 131380.000 1.793 1.793 0.000 0.0 61 G10 11998439.000 34.152 34.152 0.000 0.0 62 G11 6795279.000 19.964 19.964 0.000 0.0 63 G12 -13508.000 1.398 1.398 0.000 0.0 64 H4 6899911.000 20.249 20.249 0.000 0.0 65 H5 12336037.000 35.073 35.073 0.000 0.0 66 H6 3493238.000 10.960 10.960 0.000 0.0 67 H7 10453997.000 29.941 29.941 0.000 0.0 68 H8 485.000 1.436 1.436 0.000 0.0 69 H9 962968.000 4.061 4.061 0.000 0.0 70 H10 10256299.000 29.402 29.402 0.000 0.0 71 H11 1184423.000 4.665 4.665 0.000 0.0 72 H12 -13544.000 1.398 1.398 0.000 0.0 Group Summaries ~End Original Filename: HiSeq241_plate2_library_conc_11-19-2015; Date Last Saved: 11/19/2015 12:53:48 PM