##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 38071720 5266561 6217246 8045007 7784087 4923274 7481289 4123228 3641681 11040310 4486159 20631452 20357714 6696499 67722 9691033 7796877 9115676 764131 163562 4330293 597424 11187243 10718715 9576520 5710297 73768 10784467 72514 6487706 188061 340085 2192665 547609 7365530 5225056 37590824 7600320 58008 1970777 9999911 3233606 2511050 1243808 3149624 500745 6315954 1711448 950386 7310178 858833 7590788 1360665 1127843 8407082 250522 3694958 4401887 -12314 542966 277808 8285241 2070745 8464929 7856033 1049986 169003 1311492 1825801 1786954 -12291 259832 153799 2453087 7443256 1708389 3709271 4729629 3184041 5229032 9123497 3175726 -12328 87205 333 7065157 10133300 6280942 8385366 1004709 2731042 4427063 1791418 5423653 -12127 0 ~End Group: Standards Sample Concentration BackCalcConc Wells RFU_Values MeanRFUValue SD CV 01 100.000 98.796 A12 20631452.000 20631452.000 0.000 0.0 02 50.000 52.348 B12 10718715.000 10718715.000 0.000 0.0 03 25.000 26.607 C12 5225056.000 5225056.000 0.000 0.0 04 12.500 10.144 D12 1711448.000 1711448.000 0.000 0.0 05 6.250 4.668 E12 542966.000 542966.000 0.000 0.0 06 3.125 3.342 F12 259832.000 259832.000 0.000 0.0 07 1.563 2.533 G12 87205.000 87205.000 0.000 0.0 Group Summaries ~End Group: Unknowns_NoDiln Sample Wells RFU_Values Concentration MeanConc SD CV 01 A2 5266561.000 26.801 26.801 0.000 0.0 02 A3 6217246.000 31.256 31.256 0.000 0.0 03 A4 8045007.000 39.820 39.820 0.000 0.0 04 A5 7784087.000 38.598 38.598 0.000 0.0 05 A6 4923274.000 25.193 25.193 0.000 0.0 06 A7 7481289.000 37.179 37.179 0.000 0.0 07 A8 4123228.000 21.444 21.444 0.000 0.0 08 A9 3641681.000 19.188 19.188 0.000 0.0 09 A10 11040310.000 53.855 53.855 0.000 0.0 10 A11 4486159.000 23.145 23.145 0.000 0.0 11 B2 6696499.000 33.502 33.502 0.000 0.0 12 B3 67722.000 2.442 2.442 0.000 0.0 13 B4 9691033.000 47.533 47.533 0.000 0.0 14 B5 7796877.000 38.658 38.658 0.000 0.0 15 B6 9115676.000 44.837 44.837 0.000 0.0 16 B7 764131.000 5.705 5.705 0.000 0.0 17 B8 163562.000 2.891 2.891 0.000 0.0 18 B9 4330293.000 22.414 22.414 0.000 0.0 19 B10 597424.000 4.924 4.924 0.000 0.0 20 B11 11187243.000 54.544 54.544 0.000 0.0 21 C2 5710297.000 28.881 28.881 0.000 0.0 22 C3 73768.000 2.470 2.470 0.000 0.0 23 C4 10784467.000 52.656 52.656 0.000 0.0 24 C5 72514.000 2.464 2.464 0.000 0.0 25 C6 6487706.000 32.523 32.523 0.000 0.0 26 C7 188061.000 3.005 3.005 0.000 0.0 27 C8 340085.000 3.718 3.718 0.000 0.0 28 C9 2192665.000 12.398 12.398 0.000 0.0 29 C10 547609.000 4.690 4.690 0.000 0.0 30 C11 7365530.000 36.636 36.636 0.000 0.0 31 D2 7600320.000 37.737 37.737 0.000 0.0 32 D3 58008.000 2.396 2.396 0.000 0.0 33 D4 1970777.000 11.359 11.359 0.000 0.0 34 D5 9999911.000 48.980 48.980 0.000 0.0 35 D6 3233606.000 17.276 17.276 0.000 0.0 36 D7 2511050.000 13.890 13.890 0.000 0.0 37 D8 1243808.000 7.952 7.952 0.000 0.0 38 D9 3149624.000 16.882 16.882 0.000 0.0 39 D10 500745.000 4.471 4.471 0.000 0.0 40 D11 6315954.000 31.719 31.719 0.000 0.0 41 E2 7310178.000 36.377 36.377 0.000 0.0 42 E3 858833.000 6.148 6.148 0.000 0.0 43 E4 7590788.000 37.692 37.692 0.000 0.0 44 E5 1360665.000 8.500 8.500 0.000 0.0 45 E6 1127843.000 7.409 7.409 0.000 0.0 46 E7 8407082.000 41.517 41.517 0.000 0.0 47 E8 250522.000 3.298 3.298 0.000 0.0 48 E9 3694958.000 19.438 19.438 0.000 0.0 49 E10 4401887.000 22.750 22.750 0.000 0.0 50 E11 -12314.000 2.067 2.067 0.000 0.0 51 F2 8285241.000 40.946 40.946 0.000 0.0 52 F3 2070745.000 11.827 11.827 0.000 0.0 53 F4 8464929.000 41.788 41.788 0.000 0.0 54 F5 7856033.000 38.935 38.935 0.000 0.0 55 F6 1049986.000 7.044 7.044 0.000 0.0 56 F7 169003.000 2.916 2.916 0.000 0.0 57 F8 1311492.000 8.269 8.269 0.000 0.0 58 F9 1825801.000 10.679 10.679 0.000 0.0 59 F10 1786954.000 10.497 10.497 0.000 0.0 60 F11 -12291.000 2.067 2.067 0.000 0.0 61 G2 2453087.000 13.619 13.619 0.000 0.0 62 G3 7443256.000 37.001 37.001 0.000 0.0 63 G4 1708389.000 10.129 10.129 0.000 0.0 64 G5 3709271.000 19.505 19.505 0.000 0.0 65 G6 4729629.000 24.286 24.286 0.000 0.0 66 G7 3184041.000 17.044 17.044 0.000 0.0 67 G8 5229032.000 26.626 26.626 0.000 0.0 68 G9 9123497.000 44.874 44.874 0.000 0.0 69 G10 3175726.000 17.005 17.005 0.000 0.0 70 G11 -12328.000 2.067 2.067 0.000 0.0 71 H2 7065157.000 35.229 35.229 0.000 0.0 72 H3 10133300.000 49.605 49.605 0.000 0.0 73 H4 6280942.000 31.554 31.554 0.000 0.0 74 H5 8385366.000 41.415 41.415 0.000 0.0 75 H6 1004709.000 6.832 6.832 0.000 0.0 76 H7 2731042.000 14.921 14.921 0.000 0.0 77 H8 4427063.000 22.868 22.868 0.000 0.0 78 H9 1791418.000 10.518 10.518 0.000 0.0 79 H10 5423653.000 27.538 27.538 0.000 0.0 80 H11 -12127.000 2.067 2.067 0.000 0.0 81 A1 38071720.000 180.514 180.514 0.000 0.0 82 B1 20357714.000 97.513 97.513 0.000 0.0 83 C1 9576520.000 46.996 46.996 0.000 0.0 84 D1 37590824.000 178.261 178.261 0.000 0.0 85 E1 950386.000 6.577 6.577 0.000 0.0 86 F1 277808.000 3.426 3.426 0.000 0.0 87 G1 153799.000 2.845 2.845 0.000 0.0 88 H1 333.000 2.126 2.126 0.000 0.0 Group Summaries ~End Original Filename: HiSeq_241_plate1_LibraryConc; Date Last Saved: 11/13/2015 3:52:29 PM