##BLOCKS= 3 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 27644 31726 31018 34091 28428 31782 32753 15439 14686 14163 14413 14943 27211 24904 25522 27259 25052 27000 33471 18954 14461 13825 15171 15992 23583 25923 29944 33006 33128 31203 34760 30166 14791 16520 16024 15174 23972 24278 24679 25801 25030 26212 26637 23840 14357 14902 15523 17071 23774 26078 26421 30906 28549 30216 32513 32958 14777 15624 14977 14313 23424 24961 26741 25953 25846 27665 27912 25149 13510 14291 15029 15132 26104 24604 27268 28470 29126 29710 33228 32601 14036 14129 14404 14034 24955 25102 24736 23674 26157 28790 26922 25910 14480 14262 13226 14183 ~End Group: Unknowns Sample Wells RFU_Values Concentration Mean_Conc SD CV Dilution AdjConc 01 A1 27644.000 1.0 02 B1 27211.000 0.5 03 C1 23583.000 0.3 04 D1 23972.000 0.1 05 E1 23774.000 0.1 06 F1 23424.000 0.0 07 G1 26104.000 0.0 08 H1 24955.000 0.0 09 A2 31726.000 0.0 10 B2 24904.000 0.0 11 C2 25923.000 0.0 12 D2 24278.000 0.0 13 E2 26078.000 0.0 14 F2 24961.000 0.0 15 G2 24604.000 0.0 16 H2 25102.000 0.0 17 A3 31018.000 0.0 18 B3 25522.000 0.0 19 C3 29944.000 0.0 20 D3 24679.000 0.0 21 E3 26421.000 0.0 22 F3 26741.000 0.0 23 G3 27268.000 0.0 24 H3 24736.000 0.0 25 A4 34091.000 0.0 26 B4 27259.000 0.0 27 C4 33006.000 0.0 28 D4 25801.000 0.0 29 E4 30906.000 0.0 30 F4 25953.000 0.0 31 G4 28470.000 0.0 32 H4 23674.000 0.0 33 A5 28428.000 0.0 34 B5 25052.000 0.0 35 C5 33128.000 0.0 36 D5 25030.000 0.0 37 E5 28549.000 0.0 38 F5 25846.000 0.0 39 G5 29126.000 0.0 40 H5 26157.000 0.0 41 A6 31782.000 0.0 42 B6 27000.000 0.0 43 C6 31203.000 0.0 44 D6 26212.000 0.0 45 E6 30216.000 0.0 46 F6 27665.000 0.0 47 G6 29710.000 0.0 48 H6 28790.000 0.0 49 A7 32753.000 0.0 50 B7 33471.000 0.0 51 C7 34760.000 0.0 52 D7 26637.000 0.0 53 E7 32513.000 0.0 54 F7 27912.000 0.0 55 G7 33228.000 0.0 56 H7 26922.000 0.0 57 A8 15439.000 0.0 58 B8 18954.000 0.0 59 C8 30166.000 0.0 60 D8 23840.000 0.0 61 E8 32958.000 0.0 62 F8 25149.000 0.0 63 G8 32601.000 0.0 64 H8 25910.000 0.0 Group Summaries ~End Original Filename: NuGen_HiSeq1316_DNAConc; Date Last Saved: 6/22/2018 2:31:25 PM