DNA quantification and normalization

aáëÅä~áãÉê=mäÉ~ëÉ=kçíÉW==dÉåçãáÅëÛ=éêçíçÅçäëI=áåëíêìÅíáçåë=~åÇ=êÉÅáéÉë=~êÉ=åçí=êÉÅçããÉåÇ~íáçåëX=íÜÉó=~êÉ=àìëí=Éñ~ãéäÉë=çÑ=Üçï=óçì=Å~å= êìå= óçìê= ë~ãéäÉëK= qÜÉ= êÉÅáéÉë= ïçêâ= áå= ãçëí= Å~ëÉë= Äìí= åçí= ~ääI= ~åÇ= áí= áë= óçìê= êÉëéçåëáÄáäáíó= ~ë= ~=êÉëÉ~êÅÜÉê=íç=ÇÉÅáÇÉ=Üçï=íç=êìå=óçìê=ë~ãéäÉëLéêçàÉÅíK=DNA quantification and normalizationImportant to know about the PicoGreen ds DNAQuantitation Reagent: It is supplied as a concentrated dye solution in anhydrousdimethylsulfoxide (DMSO). Please be careful and wearnitrile gloves! It is susceptible to photodegradation so protect the workingsolution from light as much as possible. Please read about and prepare the analyzing step in the 7900 HT Sequence Detectionsystem before starting the normalization procedure.Make PicoGreen stem plates∗ Prepare an aqueous working solution of the PicoGreen reagent by making a 1:200dilution of the concentrated solution in TE buffer. It is recommended to prepare theworking solution in a plastic container as the reagent may adsorb to glass surfaces. Forbest results, this solution should be used within a few hours of its preparation.Mix for 6 stem platesPicoGreen 500µlTE buffer99.5 mlTotal100 mlBiomek FX and 7900 HT090116/CN

∗ Pour the prepared PicoGreen working solution into a BCFullReservoir. Run themethod “PicoGreen_Stampytsning_6x96_sBCFullReservoir_date” (D: > ProgramFiles > Biomek FX > Methods > Normalization), which will dispense 150µl of thesolution into each well. See figure below. Save the solution left over in a falcon tubeand put it in the -20 freezer.Transfer 1.5µl of your DNA to the PicoGreen stem plate∗ First make sure that none of your samples are empty due to evaporation. Run atransfer method in folder D: > Program Files > Biomek FX > Methods >Normalization, choose method after number of DNA plates (e.g. “Transfer_1.5µl3xDNA to PicoGreen plate”).∗ After the transfer you need to add standard curve samples to the titer plate, to be ableto calculate the concentrations of your unknown samples. Use Ceph DNA with aknown concentration of 50ng/ul to make the standard curve.Biomek FX and 7900 HT090116/CN

∗ Incubate the plates for 2 to 5 minutes at room temperature, protected from light,before the analysis on the 7900 HT Sequence Detection System.Analyze the PicoGreen plates in the 7900 HT Sequence Detection System∗ Change to the block module intended for 96 well reaction plates.∗ Make a new SDS document.Assay: allelic discriminationContainer: 96 wells clear plateMarker: PicoGreen, with the detectors SYBR and VIC. Passive reference: -none-∗ Connect to the instrument and press “Post Read”. Analyze your plate and export theresult table.Calculate the concentrations of the samples in excel∗ Open the result table exported from SDS in excel, and save it as an excel-file.∗ The PicoGreen fluorescence is most often presented in the “Allele Y Rn” column,double check this with the SDS result plot. Make a dot diagram including the knownCeph concentrations (Y-axis: PicoGreen fluorescence, X-axis: known DNAconcentration). Insert trend line and show equation.∗ Calculate with the help of the equation the sample concentration. Remember to dividethe concentration with 1,5 since this was the volume DNA transferred to thePicoGreen plate.Make an import file for normalization on Biomek FX∗ Use the template “Mall DNAConc_import file normalization” (Analysdata server >PicoGreen folder). Save it as your own and store it in your folder in Externa konsulter.Biomek FX and 7900 HT090116/CN

∗ Copy the concentration values into the template, in the column “StartConc”. Numberof decimals should be zero.∗ Save your import file on a CD-R.Run the normalization method on Biomek FX∗ Open the normalization method (D:>Program Files>Biomek FX>Methods>Normalization).∗ Import the previous created import file in “Create Data Set”:1. Browse the import file (see picture below).2. Copy the directory path and paste it in “Start” as value for “ConcentrationFile” (seepicture below).NK=mêÉëë=ÜÉêÉ=íç=ÄêçïëÉ=óçìê=áãéçêí=ÑáäÉK==2. Paste the directory path in “Start” here.Biomek FX and 7900 HT090116/CN