##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 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 25.5 -23088 -806 -17626 1685 -30549 38936026 14363492 7440277 8513341 12370895 -37736 -37660 -31443 -16019 17898 28553 -28566 19852562 19045204 14378499 20193134 38137150 -37649 -37782 -30675 5695 -2635 232545 -6997 9163834 1049982 3791528 36746742 33083276 -37799 -37740 -33133 21468 91439 7645 139420 3953329 3637664 1603815 19153566 24460428 -37716 -37743 -9295 40428 30790 -23466 -23463 782738 18385308 31181128 35368766 0 -37640 -37589 10265 8702 118028 332549 -9457 258611 12408936 12322221 38042854 -37694 -37661 -37742 13241 55225 299741 -16976 -1228 120770 9099558 6517448 39517598 -37674 -37695 -37711 13002 42409 13327 133153 7894 36799 1519253 1064198 9612455 -37728 -37679 -37695 1 2 3 4 5 6 7 8 9 10 11 12 -23088 -806 -17626 1685 -30549 38936026 14363492 7440277 8513341 12370895 -37736 -37660 -31443 -16019 17898 28553 -28566 19852562 19045204 14378499 20193134 38137150 -37649 -37782 -30675 5695 -2635 232545 -6997 9163834 1049982 3791528 36746742 33083276 -37799 -37740 -33133 21468 91439 7645 139420 3953329 3637664 1603815 19153566 24460428 -37716 -37743 -9295 40428 30790 -23466 -23463 782738 18385308 31181128 35368766 0 -37640 -37589 10265 8702 118028 332549 -9457 258611 12408936 12322221 38042854 -37694 -37661 -37742 13241 55225 299741 -16976 -1228 120770 9099558 6517448 39517598 -37674 -37695 -37711 13002 42409 13327 133153 7894 36799 1519253 1064198 9612455 -37728 -37679 -37695 ~End Group: Standards Sample Concentration BackCalcConc Wells RFU_Values MeanRFUValue SD CV 01 100.000 99.367 A6 38936026.000 38936026.000 0.000 0.0 02 50.000 51.678 B6 19852562.000 19852562.000 0.000 0.0 03 25.000 24.967 C6 9163834.000 9163834.000 0.000 0.0 04 12.500 11.946 D6 3953329.000 3953329.000 0.000 0.0 05 6.250 4.022 E6 782738.000 782738.000 0.000 0.0 06 3.125 2.713 F6 258611.000 258611.000 0.000 0.0 07 1.563 2.368 G6 120770.000 120770.000 0.000 0.0 08 0.781 2.158 H6 36799.000 36799.000 0.000 0.0 Group Summaries ~End Group: Unknowns_NoDiln Sample Wells RFU_Values Concentration MeanConc SD CV 01 A7 14363492.000 37.961 37.961 0.000 0.0 02 B7 19045204.000 49.660 49.660 0.000 0.0 03 C7 1049982.000 4.690 4.690 0.000 0.0 04 D7 3637664.000 11.157 11.157 0.000 0.0 05 E7 18385308.000 48.011 48.011 0.000 0.0 06 F7 12408936.000 33.076 33.076 0.000 0.0 07 G7 9099558.000 24.806 24.806 0.000 0.0 08 H7 1519253.000 5.863 5.863 0.000 0.0 09 A8 7440277.000 20.660 20.660 0.000 0.0 10 B8 14378499.000 37.998 37.998 0.000 0.0 11 C8 3791528.000 11.541 11.541 0.000 0.0 12 D8 1603815.000 6.074 6.074 0.000 0.0 13 E8 31181128.000 79.988 79.988 0.000 0.0 14 F8 12322221.000 32.860 32.860 0.000 0.0 15 G8 6517448.000 18.353 18.353 0.000 0.0 16 H8 1064198.000 4.726 4.726 0.000 0.0 17 A9 8513341.000 23.341 23.341 0.000 0.0 18 B9 20193134.000 52.529 52.529 0.000 0.0 19 C9 36746742.000 93.896 93.896 0.000 0.0 20 D9 19153566.000 49.931 49.931 0.000 0.0 21 E9 35368766.000 90.453 90.453 0.000 0.0 22 F9 38042854.000 97.135 97.135 0.000 0.0 23 G9 39517598.000 100.821 100.821 0.000 0.0 24 H9 9612455.000 26.088 26.088 0.000 0.0 25 A10 12370895.000 32.981 32.981 0.000 0.0 26 B10 38137150.000 97.371 97.371 0.000 0.0 27 C10 33083276.000 84.741 84.741 0.000 0.0 28 D10 24460428.000 63.193 63.193 0.000 0.0 Group Summaries ~End Original Filename: HiSeq224; Date Last Saved: 10/5/2015 1:45:01 PM