##BLOCKS= 5 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) v 1.3; Excitation changed to 485 nm and Emission changed to 535 nm. ~End Plate: Standards 1.3 PlateFormat Endpoint Fluorescence FALSE Raw FALSE 1 1 535 1 12 96 485 Manual 0 1 8 Temperature(¡C) 1 2 3 4 5 6 7 8 9 10 11 12 29 24452390 27408884 -6348 -6277 -6341 -6428 -6388 -6400 -6405 -6354 -6391 -6238 13704656 16541589 -6411 -6327 -6414 -6320 -6371 -6468 -6448 -6320 -6324 -6411 7242121 8538253 -6308 -6311 -6367 -6397 -6306 -6303 -6429 -6323 -6328 -6350 3386490 4265889 -6302 -6334 -6305 -6309 -6292 -6373 -6292 -6378 -6351 -6459 1730805 2170023 -6342 -6421 -6340 -6355 -6347 -6343 -6380 -6372 -6348 -6392 939560 1086969 -6349 -6334 -6322 -6356 -6371 -6380 -6383 -6338 -6390 -6481 485129 541431 -6381 -6315 -6386 -6284 -6352 -6370 -6134 -6389 -6394 -6365 -1139 1139 -6329 -6366 -6332 -6351 -6383 -6393 -6439 -6385 -6346 -6454 ~End Plate: Plate1 1.3 PlateFormat Endpoint Fluorescence FALSE Raw FALSE 1 1 535 1 12 96 485 Manual 0 1 8 Temperature(¡C) 1 2 3 4 5 6 7 8 9 10 11 12 29 1789834 2372628 1196925 781309 575497 3822919 509401 1128781 1309184 423208 1205729 2537139 1477818 5131094 372058 352932 582718 2059707 363356 1754429 449057 564871 807831 52506 1262624 1372606 762205 872035 325368 1682834 679625 771091 221123 224523 1294179 178658 1058569 879201 1298944 430675 2245355 1518399 742589 839405 205405 6880206 1841152 862980 1535046 341547 314581 1020281 2031398 462591 2637071 456316 253765 4866059 1412458 1348347 723296 1581347 741424 2121568 471155 2052377 1331802 782199 389917 161486 478378 1297220 2869297 1673981 478287 3548649 419065 734538 498791 630760 127781 1602227 502910 348005 1305316 400563 464526 4103165 313735 425483 1157161 666142 212216 644633 1816457 189642 ~End Group: Stand Sample Well Concentration ng Values MeanValue Std.Dev. CV% 01 A2 117.000 Masked Masked Masked Masked 02 B2 50.000 16541589.000 16541589.000 0.000 0.000 03 C2 25.000 8538253.000 8538253.000 0.000 0.000 04 D2 12.500 4265889.000 4265889.000 0.000 0.000 05 E2 6.250 2170023.000 2170023.000 0.000 0.000 06 F2 3.125 1086969.000 1086969.000 0.000 0.000 07 G2 1.563 541431.000 541431.000 0.000 0.000 Group Summaries ~End Group: Unknowns Sample Wells RFU_Values Concentration Mean_Conc SD CV Dilution AdjConc 01 A1 1789834.000 5.113 3.291 3.491 106.1 0.0 0.000 A2 2372628.000 6.875 A3 1196925.000 3.319 A4 781309.000 2.062 A5 575497.000 1.440 A6 3822919.000 11.261 A7 509401.000 1.240 A8 1128781.000 3.113 A9 1309184.000 3.659 A10 423208.000 0.979 A11 1205729.000 3.346 A12 2537139.000 7.373 B1 1477818.000 4.169 B2 5131094.000 15.218 B3 372058.000 0.825 B4 352932.000 0.767 B5 582718.000 1.462 B6 2059707.000 5.929 B7 363356.000 0.798 B8 1754429.000 5.006 B9 449057.000 1.058 B10 564871.000 1.408 B11 807831.000 2.143 B12 52506.000 -0.142 C1 1262624.000 3.518 C2 1372606.000 3.851 C3 762205.000 2.005 C4 872035.000 2.337 C5 325368.000 0.684 C6 1682834.000 4.789 C7 679625.000 1.755 C8 771091.000 2.032 C9 221123.000 0.368 C10 224523.000 0.379 C11 1294179.000 3.614 C12 178658.000 0.240 D1 1058569.000 2.901 D2 879201.000 2.359 D3 1298944.000 3.628 D4 430675.000 1.002 D5 2245355.000 6.490 D6 1518399.000 4.292 D7 742589.000 1.945 D8 839405.000 2.238 D9 205405.000 0.321 D10 6880206.000 20.508 D11 1841152.000 5.268 D12 862980.000 2.309 E1 1535046.000 4.342 E2 341547.000 0.732 E3 314581.000 0.651 E4 1020281.000 2.785 E5 2031398.000 5.843 E6 462591.000 1.099 E7 2637071.000 7.675 E8 456316.000 1.080 E9 253765.000 0.467 E10 4866059.000 14.416 E11 1412458.000 3.971 E12 1348347.000 3.777 F1 723296.000 1.887 F2 1581347.000 4.482 F3 741424.000 1.942 F4 2121568.000 6.116 F5 471155.000 1.124 F6 2052377.000 5.907 F7 1331802.000 3.727 F8 782199.000 2.065 F9 389917.000 0.879 F10 161486.000 0.188 F11 478378.000 1.146 F12 1297220.000 3.623 G1 2869297.000 8.377 G2 1673981.000 4.762 G3 478287.000 1.146 G4 3548649.000 10.432 G5 419065.000 0.967 G6 734538.000 1.921 G7 498791.000 1.208 G8 630760.000 1.607 G9 127781.000 0.086 G10 1602227.000 4.545 G11 502910.000 1.220 G12 348005.000 0.752 H1 1305316.000 3.647 H2 400563.000 0.911 H3 464526.000 1.104 H4 4103165.000 12.109 H5 313735.000 0.648 H6 425483.000 0.986 H7 1157161.000 3.199 H8 666142.000 1.714 H9 212216.000 0.341 H10 644633.000 1.649 H11 1816457.000 5.193 H12 189642.000 0.273 Group Summaries ~End Original Filename: 141217_CBA_Other002; Date Last Saved: 12/17/2014 4:56:10 PM