7900HT Fast Real-Time PCR System with Fast 96-Well Block Module

The threshold is the numerical value assigned for each run that reflects the average dRn (change in fluorescence) during the initial cycles of PCR (baseline). The threshold is set by determining a statistically significant point (or value) above the baseline. You can manually adjust the threshold should you desire, to the place in the geometric phase where your replicates are tightest. For more information on how to manually set a threshold, please refer to the tutorial entitled Data Analysis on the ABI PRISM 7700: Setting Baselines and Thresholds (P/N 4370923). While this tutorial is based on the ABI PRISM 7700 system, the concepts are still valid for the Applied Biosystems real-time PCR instruments. You can search the document on our website by using the part number above as the keyword.

If you choose not to manually adjust the baselines and thresholds, the SDS software on the Applied Biosystems real-time PCR instruments has an Auto Ct algorithm that can be used to automatically generate baseline and threshold values for individual detectors. The algorithm calculates baseline and threshold parameters for a detector based on the assumption that the data exhibits the "typical" amplification curve. Experimental error (i.e. contamination, pipetting inaccuracies) can produce amplification curves that deviate significantly from a typical amplification curve. The data from these irregularities can affect the Auto Ct algorithm by causing it to generate incorrect baseline and threshold parameters for the associated detector. Therefore, Applied Biosystems recommends that after analysis of experimental data, you review all baseline and threshold parameters determined by the Auto Ct algorithm.

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You have to run a calibration when a new sample block format is used in the 7900HT Fast Real-Time PCR System, but it is not necessary to run the pure dye calibration if the block has been used before. For example, if you use a 384-well sample block for the first time, then you have to run a 384-well block calibration. Later, should you switch to a 96-well block and then switch back to a 384-well block again, then there is no need to rerun the calibration of the 384-well sample block. We recommend that you run a background run each time you exchange the sample block because dust particles or other contaminants could get into the wells while the block is being stored outside of the instrument. Please consult the 7900HT User Manual, Cat. No. 4317596, for the recommended maintenance procedures.

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The log file tracks events during the course of an instrument run. To locate the log file, open up the hard drive of the instrument computer. Open the Applied Biosystems folder, then the SDS 2.x.x folder, then the log folder. The log file is a Microsoft Word file in .doc format and is saved as the date the run was initiated.

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To program a long-term hold on the Applied Biosystems 7900HT Fast Real-Time PCR System, launch the SDS software and open a new plate. Click on the Instrument tab. Move the cursor to the end of the thermal cycling profile, below the time box, and click. This will create a solid vertical black line. Click on “Add Hold”. Change the temperature to 4.0 and the time to the highest possible time, 98:59. Although an infinity symbol will not appear, the instrument will run for 98 hours and 59 seconds. The run must be aborted when you want to analyze the data.

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While the Applied Biosystems 7900HT Fast Real-Time PCR System is idle, it puts off 6600 BTU/hour. While it is cycling, it puts off 8800 BTU/hour.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.