TITLE: DNA quantification in plates (picogreen protocol)
AUTHORS: Jenn Coughlan, Jen Modliszewski, Cathy Rushworth

[STEPS]
?. [Sample Prep + Standard Curve]
Allow the Quant-iT PicoGreen reagent to warm to room temperature before opening the vial. PicoGreen is light sensitive; while thawing, wrap in aluminum foil, stick in desk drawer. Note: PicoGreen takes a long time to thaw—maybe an hour.
?. [Sample Prep + Standard Curve]
Prepare a 1x working solution of TE using 20x TE (supplied) and sterile di H2O.
?. [Sample Prep + Standard Curve]
Prepare a working solution of PicoGreen by doing a 1:200 dilution (PicoGreen: final volume). Prepare solution in plastic falcon tube wrapped in aluminum foil, as the reagent is light sensitive. Do not use glass as PicoGreen may adsorb to glass.You need 48ul of total solution per sample. Make extra for errors.
?. [Sample Prep + Standard Curve]
Prepare standard curve using Lambda DNA standard (supplied at a concentration of 100ng/ul or 100ug/ml, comes with PicoGreen) and 1x TE in 8 tubes, as below. Load 2ul from each tube into one column (A12-H12). You will need to increase these volumes if running >2 plates.Tube #                Contents                                 Concentration        Final volume1               10 ul TE;  10 ul Lambda DNA            50.0 ng/ul                 10 ul2              10 ul TE;   10 ul from tube                 125.0 ng/ul                10 ul3              10 ul TE;   10 ul from tube                 212.5 ng/ul                8 ul4              4 ul TE;      6 ul from tube                   37.5 ng/ul                 10 ul5              6 ul TE;      4 ul from tube                   35.0 ng/ul                  9 ul6              8 ul TE;      2 ul from tube                 32.5 ng/ul                  10 ul7              4 ul TE;      1 ul from tube                 51.0 ng/ul                    5 ul8              10 ul TE                                                    0.0 ng/ul               10 ul
?. [Sample Prep + Standard Curve]
Pipet 2ul sample into wells in Costar black microplate (#3915), with the standard curve in wells A1-H1. Use one column of wells for a standard curve in each plate. Pipet your samples into the center of each well. If your DNA is in tubes, this process will take a long time, and you risk dehydration of the samples. You will need to work quickly, and cover or seal plates somehow after finishing one.
?. [Sample Prep + Standard Curve]
THE REMAINING STEPS SHOULD DONE BY A PLATE READER- You will need:Reservoir and PicoGreen working solutionP200 multichannel pipetAs many tips as you have plates/samplesWaste containerUSB driveSamples in Costar plates (stack and use lid for top one)Aluminum foil covers + piece of aluminum foilProtocolWriting utensilGloves
?. [Plate Reader]
Pour PicoGreen solution into reservoir and pipet 48 ul PicoGreen working solution into each well. Cover plate with aluminum foil covered Rainin lid. Cover reservoir with aluminum foil. Tip plate slightly and tap sides of plate gently but firmly onto bench to get liquid to cover the bottom of every well. This is difficult, but these wells have a minimum recommended volume of 75ul, and the liquid really should cover the bottom of the well to ensure an accurate reading. You will need to tap harder than you think, but be careful!
?. [Plate Reader]
. Incubate for 2-5 minutes, keeping foil cover on to protect from light.
?. [Plate Reader]
Read your samples on the plate reader, be sure to follow its specifications. Below is the settings used for the Willis lab:Protocol settings for Willis_DNAquant50 protocolPlate layout: 96 well, read all of wellsExcitation: 485 nmEmission: 530 nmRead from topEndpoint readingDetect fluorescenceSensitivity (= Gain): 50
?. [Calculating DNA concentrations]
Plot your standard curve DNA concentrations in ng/ul (x) and fluorescence scores (y) as a scatter plot.y = mx + bIn Excel:Select a data point. Go to Chart--> Add Trendline. The line should be linear, and check the “display equation on chart” option, and the R2 to make sure you didn’t really mess up the standard curve, although that should be apparent from the line on the chart.Solve for x to determine the concentration of your samples.