Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

Workflow OverviewWorkflow OverviewExperiments/Runs Performedon the 7900HTInstrumentAbsoluteQuantificationWorkflowAbsolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16Dissociation Curve (Melting Curve) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6Pure Dye (Spectral Calibration) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-20Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6RNase P Instrument Performance Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-301. Create an absolute quantification plate document (see page 3-9).2. * Apply detectors to the plate document:a. Create detectors for the absolute quantification probes (see page 3-11).b. Copy the detectors to the plate document (see page 3-13).3. Configure the plate document with tasks and quantities:a. Configure the plate document with detector tasks (NTC, Standard, andUnknown) (see page 3-16).b. Assign quantities to the wells of the plate document that contain standards(see page 3-17).4. Program the method for the absolute quantification run (see page 3-19).5. If performing an assay in which you would like to collect dissociation data, add atemperature ramp to the thermal profile to perform a dissociation curve analysis(see page 3-23).6. Choose from the following:– If running a single plate, then continue to step 2.– If running the first plate in a series of plates with identical assayconfigurations, then save the plate document as a template (see page 3-24).7. Create a plate document from the template created in step 2 (see page 3-26).8. Configure the document with sample names and plate information (seepage 3-28).9. Prepare and run the absolute quantification plate or plates (see page 4-1).10.Analyze the run data (see page 6-5).*Steps 2 and 2 can be eliminated by importing the plate document setup informationfrom a tab-delimited text file. See “Importing Plate Document Setup Table Files”on page A-2 for more information.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-5DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm