RealTimePCROperationProcedure.doc

Solexa Sequencing Library Preparation Real Time PCR Experiment Operation ProcedureDocument Serial No.: _Version No.: _Creator: _Date: _Examined by: _Authorized by: _Effective Date: _Distribution No.: _ BGI- Hong Kong Co., Limited Genomics Sequencing PlatformContentApplicability and Limit3Abstract3Abbreviations4Materials and Methods61 Reagents and Consumables81.1 Reagents81.2 Consumambles92 Equipment93 Experiment procedures93.1 Prepare the standard dilution series103.2 Dilution of samples103.3 Prepare the reaction mix133.4 Prepare the reaction plate133.5 Prepare the run143.6 Data Analysis134 Data management and Records management14 5 Quality Assessment/ Quality Control16References17Applicability and LimitIn this experiment apply to the use StepOnePlusRealTime-PCR amplification of the DNA PE, SE library and the transcriptome, expression profiling, small RNA library preparation group for the precise quantification before on the cluster station.AbstractQuantitative PCR experiments based on Agilent 2100 Bioanalyzer results oftests on the library as a reference. The main procedure included: 1. the standard dilution series. 2. Dilution of samples. 3. Prepare Reaction Mix. 4. Prepare the reaction plate. 5. Set for the Run. 6. Run the experiment. 7. Data analysis. All procedure needs 3 to 4 hours. AbbreviationsBaselineIn the amplification plots, a line fit to the fluorescence levels during the initial stage of PCR, when there is little change in fluorescence signal.Ct ValueThe PCR cycle number at which the fluorescence meets the threshold in the amplification plot.ThresholdIn amplification plots, the level of fluorescence above the baseline and within the exponential growth region. Materials & Method1. Reagents and Consumables1.1ReagentsReagents NameLot. No.Factory10X Ex Taq BufferAA6901ATaKaRadNTP mixtureBG4401ATaKaRaMgSO0010805NEW England BioLABSROX Reference Dye761819InvitrogenPE Primer 1.1M165842SangonPE Primer 2.1M100658SangonSE Primer 1.1M76070SangonSE Primer 2.1M67154SangonNGEX Primer 1.1 M142353SangonNGEX Primer 2.1M142354SangonEva green 9E0713BiotiumTaqman ProbeSangonEX Taq HSCN6501AASangonDMSOSangon BetaineSigmaTween 20X BufferSigma1.2 ConsumablesConsumablesLot. No. Factory2.0ml microtubes100118-262Axygen1.5ml microtubes100612-041Axygen0.6ml microtubes090704-301Axygen0.2ml PCR tubes090420-179AxygenMicroAmp Optical 96-Well Adhesive Film4311971Applied Biosystems48-well reaction plates96-well reaction plates96-well Film2. EquipmentsEquipmentsLot. No.FactoryStepOne Plus Real-Time PCR SystemApplied BiosystemsVortexQilinbeierMini centrifuge10D008BEZ FUGE-20 Fridge4 Fridge3. Experiment Procedure3.1 Prepare the Standard Dilution SeriesStandard CurveSource Volume (L)Diluent Volume (L)Total Volume (L)Concentration (pM)Std11 l (10nM)9101000Std22 l (Std1)1820100Std32 l (Std2)182010Std42 l (Std3)18201Std52 l (Std4)18200.1Figure 3-1 Standard Dilution Series1. Labeled a separate 0.2ml PCR microcentrifuge tube for each Standard: Std 1, Std 2, Std 3, Std 4 and Std 5.2. Added the required volume of Milli-Q water (diluents) to each empty tube:TubeVolume of Diluent to Add (L)Std 19Std 218Std 318Std 418Std 518Figure 3-2 Volume of Diluent3. Vortex the Standard for 10 to 15 seconds, then centrifuge the tube briefly.4. Added 1 l of Standard to the Std 1 tube by using a new pipette tip.5. Vortex Std 1 for 10 to 15 seconds, then centrifuge the tube briefly.6. Added 2 l of dilution 1 to the Std 2 tube by using a new pipette tip.7. Vortex Std 2 for 10 to 15 seconds, then centrifuge the tube briefly.8. Repeated the step 6 and 7 by Std 2 to Std 3, Std 3 to Std 4 and Std 4 to Std 5.9. Placed the Standards on ice cooler until the reaction plate prepared.Preparation Guidelines/Precaution: Standards are critical for accurate analysis of run data. Any mistakes or inaccuracies in making the dilution directly affect the quality of results. The quality of pipettors and tips and the care used in measuring and mixing dilutions affect accuracy. Use TE buffer or water to dilute the Standards. Pipetting up and down is recommended in each step.3.2 Dilution of Samples1. Labeled the sample name on a separate 0.6ml microcentrifuge tube for each diluted sample.2. Added the required volume of Milli-Q water (diluent) to each empty tube:TubeSample Volume (L)Diluent Volume (L)Sample name199Figure 3-3 Volume of Diluted Sample3. Added 1 l of sample to each tube and vortex each diluted sample for 10 to 15 seconds, then centrifuge the tubes briefly.4. Placed the Standards on ice cooler until the reaction plate prepared.Preparation Guidelines/Precaution: Sample dilutions may be necessary because the sample volume is limited to 10% of the total reaction volume in the StepOne Software. The sample must perform dilutions before adding the samples to the final reaction mix. Use TE buffer or water to dilute the samples. Pipetting up and down is recommended in each step.3.3 Prepare the Reaction Mix1. Labeled a 2.0ml microtube and covered an aluminum foil for the master mix: PE MIX.2. Added the required volumes of each reagent to the tube:ReagentsReaction Volume (1X) (l)Reaction Volume (50X) (l)10X Taq Buffer1 l50 lMgSO0.1 l5 lTween 20X0.1 l5 lDMSO0.5 l25 lH2O3.35 l167.5 lBetaine2 l100 lFigure 3-4 Master Mix3. Mix the reaction mix gently by pipetting up and down, then cap the tube.4. Centrifuge the tube briefly to remove air bubbles.5. Labeled a 1.5ml microtube for the Reaction Mix: reaction mix.6. Added the required volumes of each reagent to the tube:ReagentsReaction Volume (1X) (l)Reaction Volume (37X) (l)PE Mix7.05 l260.85 ldNTP0.8 l29.6 lProbe0.25 l9.25 lROX0.2 l7.4 lPrimer 1.10.3 l11.1 lPrimer 2.10.3 l11.1 lEX Taq HS0.1 l3.7 lFigure 3-5 Reaction Mix7. Place the reaction mix on ice until the reaction plate prepared.Preparation Guidelines/Precaution: Mask and glove should be wearing before experiment and 70% alcohol should be used to clean up the bench. If the experiment includes more than one target assay, prepare the reaction mix for each target assay separately. Keep the reaction mix protected from light. Excessive exposure to light may affect the fluorescent probes. Mix the master mix thoroughly by swirling the bottle. Thaw any frozen samples by placing them on ice. When thawed, resuspend the samples by vortexing, then centrifuge the tubes briefly. Pipet up and down 3 times when adding Taq enzyme into the reagents.3.4 Prepare the Reaction Plate1. Put a 48-well reaction plate or a 96-well reaction plate on the support base.2. Added 9 l of Reaction Mix to the appropriate wells in the reaction plate.3. Added 1 l of Standard to the appropriate wells in the reaction plate. ( Std 1, Std 2 and Std 3 have two replicates and Std 4 and Std 5 have one replicate only)4. Added 1 l of Sample to the appropriate wells in the reaction plate. (Every samples have three replicates)5. Added 1 l of Water to the appropriate wells in the reaction plate for negative control reactions.6. Sealed the reaction plate with optical adhesive film.7. Centrifuge the reaction plate with 4000rpm in 2mins to remove the air bubbles.8. Confirm that the liquid is at the bottom of each well of the reaction plate. If not, centrifuge the reaction plate again at a higher speed and for a longer period of time.9. Placed the reaction plate on ice in the dark until the run performed. Preparation Guidelines/Precaution: Make sure used the appropriate consumables. Do not allow the bottom of the reaction plate to become dirty. Fluids and other contaminants that adhere to the bottom of the reaction plate can contaminate the sample block and cause an abnormally high background signal.3.5 Prepare for the Run1. Double-click StepOne Software shortcut.2. Click Advanced Setup to create an experiment.3. Click the Experiment Name field, then enter Name, Date and Experiment Method.4. Select StepOnePlus Instrument (96 Wells).5. Select Quantitation for the experiment type.6. Select Standard Curve as the quantitation method.7. Select TaqMan Reagents or SYBR GREEN for the reagents.8. Select Standard (2 hours to complete a run) for the ramp speed.9. Select gDNA (genomic DNA) for the template type.10. Click Plate Setup on the Navigation pane.11. Select Define Targets and Samples, then enter Target name. (If Taqman is used, Reporter set FAM and Quencher set NFQ-MGB. If SYBR is used, Reporter set FAM and Quencher set NONE.) Leave the default in the color field.12. Select Define Samples and enter Samples name. Leave the default in the color field.13. Click Assign Targets and Samples and select wells in the view plate layout.14. Click Assign Targets to the selected wells (S= Standard, U= unknown Sample and N= Negative Control)15. Click Assign Samples to the selected wells.16. Click Run method on the Navigation pane.17. Select Reaction Volume Per well and enter the reaction volume 10 l.18. Select the temperature and time as follow:Holding Stage502minutes9510secondsCycling Stage9430seconds35cycles6530seconds7245secondsFigure 3-6 DNA PE & INDEX Run MethodHolding Stage9510secondsCycling Stage9515seconds35 cycles6530seconds7230secondsMelting Curve StageFigure 3-7 DNA SE Run Method3.6 Data Analysis1. Click the analysis in the left navigation pane at the end of the experiment.2. Click the Amplification Plot to view the amplification curve.3. Click the Standard Curve to view the standard curve of R2 value, slope and amplification efficiency. 4. Click the View well table to view the Ct value and concentration.5. Export data to excel sheet by click on File, select the File menu in the Export, the pop-up dialog box in the Export Properties in check Results, and then Set the Export File Name, and Export File Location.6. Click on Start Export.After export the data in the pop-up dialog box, select Close Export Tool.7. View the data, due to the concentration range of standard 0.1pM-1nM, in order to ensure the credibility of the data should be removed beyond the scope of the standard curve of samples with a concentration gradient.Dilute the sample are required in accordance with the dilution of the original concentration and then converted to the average demand.4. Data Management and Records ManagementThis ex