7900HT Fast Real-Time PCR System Support -Getting Started

The calibration plates can be stored and reused three times for up to 12 months after you first open them, so make sure to return them to their original packaging and return them to –20°C storage until the next use. (Note: Due to the small volumes, calibration array cards cannot be reused. Please discard them after use.) If needed, you can make your own background plate using deionized water.

The RNaseP verification plate contains template, master mix, and a TaqMan assay for RNaseP. It is used to verify that the instrument is performing to specifications. If you have reason to suspect there is something wrong with the instrument, or if you want to rule out a chemistry issue, the RNaseP plate is a good way to test the system. The RNaseP verification plate is a single-use plate.

Follow the directions in the 7900HT Fast Real-Time PCR System Maintenance Guide, Chapter 7, to decontaminate the sample block.

Do not use MicroAmp Optical Caps or MicroAmp Optical Tubes with the 7900HT Fast Real-Time PCR System. The instrument is not designed to run MicroAmp Tubes. Please be aware that use of tubes may damage its internal components.

The 7900HT Fast Real-Time PCR System can be used to run assays designed with custom dyes (dyes not manufactured by Life Technologies). Custom dyes must fluoresce within the 500–660 nm spectral range measured by the 7900HT instrument. The calibration is done in two steps. In the first step, you will need to obtain an oligo labeled with your dye (no quencher), and then create a plate containing different concentrations of the oligo (~25–3,200 nM). This is done to determine the optimal concentration to use for the actual calibration. In the second step, you will create a plate of the oligo at the concentration determined in the previous step and then run a custom dye calibration. You need to fill at least 3 full columns for adequate data collection. Follow the directions in Appendix B of the 7900HT Fast Real-Time PCR System Maintenance Guide for more specific instructions.

Using the instrument computer when a run is in progress is not recommended, as this poses a risk of corrupting the data.

The following volumes are supported for each 7900HT instrument block:

• Standard 96-well block: 20–50 µL
• Fast 96-well block: 10–30 µL
• 384-well block: 5–20 µL
• TaqMan Low Density Array block: ~1µL

No. On the 7900HT Fast Real-Time PCR System, the software will use the last data collection step in the cycling stage for all amplification plots and Ct analysis. So even if you were to set two separate steps with ‘Data Collection On’ you would only be able to view and analyze data from the latter step.

On the 7900HT Fast Real-Time PCR System, run files will be saved to a default folder on the connected computer, unless you change it.

To find or change the default folder, go to Tools → Options → General. Here you will see a Data Folder and an Import Folder. The default location is shown. If you want files to be saved to (or open from) a different location, click ‘Browse’ and choose the new folder.

Yes, but the data is not compatible with our Protein Thermal Shift™ software. Below are instructions on how to program the software and a reference for the analysis.

1. Create a new experiment using File → New in the 7900HT software. Click ‘OK’.
2. Go to the bottom of the Setup panel, select Passive Reference as None.
3. Click on ‘Add Detector’ button on the setup panel.
   • If you do not have a preset detector,click on ‘New’ from Detector Manager window, so you can define a new detector. Make sure the reporter dye is ROX™, and quencher is Non Fluorescent.
   • Once you have the desired detector highlighted in the Detector Manager window, click the ‘Copy to Plate Document’ button, then click ‘Done’. You will see the detector now in the Setup panel.
4. Next go to Instrument tab, in which you will see the default the thermal profile.
5. Click at the end of the thermal profile, a vertical line will appear.
6. Click the ‘Add Dissociation Stage’ button, then highlight and delete the PCR stages.
7. Edit the profile as below, to go from 25°C (2 min) to 95°C (15 sec).
8. Click on the ‘Ramp Rate’ tab, and change the ramping to 1%.
9. Save the file, and start the run (or save as a template).

Please note that you will have to perform the data analysis separately from the raw data. You can refer to this reference as a starting point (Nat Protoc 2007;2(9):2212-21).

In the 7900HT SDS software, you can run a melt curve at the end of an Absolute Quantification plate run, or run a melt curve by itself. You cannot directly add a melt curve to the end of a Relative Quantification (or ddCT plate).

For the former, here are the steps:

• File → New, Assay: Standard Curve (AQ), Click ‘OK’.
• In the new document, click on the ‘Instrument’ tab (right hand side). You should now see the thermal profile.
• Click in the box at the last step such that a black line appears. Click on the ‘Add Dissociation Stage’ button which should now be active.
  

If you just want to run a melt curve (no amplification stage), then you will want to highlight the other steps (click and drag across Stages 1–3). Then click on the ‘Delete Step’ button. Now you can run just the melt curve.

If you want to run a melt curve with a RQ file, you have two options. One, if you have the v2.4 software, you can run the file as demonstrated above as AQ with a melt curve, and then use the conversion tool later to switch to an RQ file. The other option is to run the file as RQ, and then once the run is complete, set up the melt curve run as described above.

Detectors can be added to a plate before or after a run is complete. To do so:

• Go to Tools → Detector Manager.
• Find the Detector that you want to add from the list, and select it by clicking on it.
• Click on ‘Copy to Plate Document’. (If you have more than one detector to add, repeat these steps.)
• When finished, click “Done”. You should now see the Detectors you added under the Setup Tab.
• Now highlight the wells that you want and check the box next to the detector you want to use. Repeat for however many detectors you need. Analyze the plate by clicking on the Green Arrow and then ‘Save As’ a new file name.

The Applied Biosystems 7900HT Fast Real-Time PCR Systems use the following dye sets for calibration: FAM™, SYBR, VIC, ROX™, NED™, TAMRA™, JOE™, TET™ dyes. Custom dyes can also be used that read between 500 nm and 660 nm, although you will have to calibrate the system first for any new dye.

For genotyping data, we recommend TaqMan Genotyper Software. For relative quantitation, we recommend ExpressionSuite™ Software.