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Calibration

Recommended Maintenance Schedule
Power on/off the computer controlling the instrument Weekly
Check computer disk space. If necessary, archive or back up your experiment files and instrument settings Weekly
Background calibration Every month
Run disk cleanup and disk defragmentation Every month
Perform an instrument self test Every month
Pure dye calibrations Every 6 months
ROI calibration Every 6 months
Optical calibration Every 6 months
RNaseP instrument verification After the instrument has been moved, or as needed to verify instrument performance

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. If needed, you can make your own background plate using deionized water. Please follow the directions in the 7500 and 7500 Fast Real-Time PCR System Maintenance Guide (Appendix C) for more details.

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, if the instrument has been moved, 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.

Operation
Instrument Specifications

Block Options
(Interchangeable)

7500:  96-well (standard)
7500F: 96-well (Fast)

Sensitivity Down to 1 copy
Dynamic Range 9 logs of linear dynamic range
Calibrated Dyes FAM™, SYBR®, VIC®, ROX™, NED™, TAMRA™ dyes (Cy®3, Cy®5, and Texas Red® dyes – 7500F only)
Detection Method SYBR® dye, primer-probe detection
Resolution Detect changes as little as 1.5-fold
Reaction Volume Range

20–100 µL (7500)
10–30 µL  (7500F)

Reaction Speed Fast or standard (7500 has standard mode only)
Optics Tungsten-halogen lamp, 5 excitation filters, 5 emission filters, CCD Camera
Temperature Range 4–99.9°C
Run Time

<2 hr (standard mode)
~35 min (Fast mode – 7500F only)

Regulatory Statement For Research Use Only. Not for use in diagnostic procedures.
Temperature Accuracy ±0.25°C (between 35°C and 95°C, after 3 min)
Temperature Uniformity ±0.5°C (after 30 sec)
Thermal Cycling System Peltier-based system
Available Applications Gene expression, genotyping, copy number variation, HRM, protein thermal shift, protein detection, mutation detection, miRNA, presence/absence
Dimensions 34 cm (W) x 45 cm (D) x 49 cm (H)
Weight 34 kg (75 lb)
Remote Monitoring No
On-Board Memory No
Setup Configurations PC-controlled only

The Applied Biosystems® 7500 and 7500 Fast Real-Time PCR Systems use the following dye sets for calibration: Cy®3, Cy®5, FAM™, JOE™, NED™, ROX™, SYBR® Green, TAMRA™, Texas Red®, and VIC® dyes. The following figure shows the emission spectrum for each dye, and the filters and wavelengths at which each dye is read. Custom dyes that are read between 520 and 650 nm can also be used, although you will have to calibrate the system first for any new dye.

7500 Dye

The 7500 and 7500 Fast Real-Time PCR Systems can be used to run assays designed with custom dyes (dyes not manufactured by Life Technologies). Custom dyes must fluoresce within the 520–650 nm spectral range measured by the 7500 or 7500 Fast instrument. To use a custom dye, you must first determine what the right concentration of the dye is. You will need to order an oligo with a 5’ custom dye but no quencher. Make up a plate with different concentrations of this oligo (~25–3,200 nM) and use the ROI Inspector to assess fluorescence. Choose the concentration that displays the brightest possible signal without saturation in all filters. Once you have found the correct concentration, create a full plate of custom dye at this concentration and perform the custom dye calibration. See Appendix B in the 7500 and 7500 Fast Real-Time PCR System Maintenance Guide for full details.

7500 Custom Dye Saturation

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 instrument block:

  • 7500: 20–100 µL reactions
  • 7500 Fast: 10–30 µL reactions

No. 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 analysis data from the latter step.

Run files will be saved to a default folder on the connected computer, unless you change it. To find or change the default folder (in SDS v2.0.1 or later), go to Tools → Preferences → Defaults. 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.

In a touchdown PCR experiment, you will either change the temperature or the time of a particular PCR step with every cycle. Most commonly, the annealing temperature is adjusted throughout the experiment, such that the specificity is increased in the early cycles and the efficiency in the later cycles.

In this example, we will set the method to do the following:

Formula
  1. Go to File → New Experiment → Advanced Setup. Fill out the relevant options as you normally would. Touchdown 1

  2. Go to the Run Method under the ‘Setup’ section and you should see the Graphical View of your thermal profile. Check the box next to ‘Enable AutoDelta’. You should see some grey triangles appear next to the Temperature and Time at every step in the Cycling Stage. (Note: If you want to start the changes at a later cycle, set this here under ‘Starting Cycles’.)
  3. A new window called ‘AutoDelta Settings’ will open up. Select the appropriate options. In this example we are decreasing the temperature by 0.4°C per cycle, so choose (“-“) and (0.40). Click ‘Save Setting’. You will then see a green triangle show up next to the parameter you changed, in this case next to the 72°C step. Your new method has now been applied. Touchdown 3

Yes. If you have the newer version of the software (v2.0.1 or later), which creates *.eds files, your data will be directly compatible with our Protein Thermal Shift™ Software. If you have the older software (v 1.x), you will have to program the software differently (see below) and analyze the results independently. For more details on the analysis, you can refer to this paper: “The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.” Nat Protoc 2007;2(9):2212-21.

For an experiment using SDS v1.x, follow the directions below:

  1. Create a new experiment, choosing Absolute Quantification assay type. Click ‘Next’.
  2. Go to ‘Select Detectors’. If there is no predefined detector for this application, create a new one. Make sure the Reporter Dye is ROX, and Quencher Dye is none.
  3. Assign the detector to the plate. Select none for the Passive Reference.7500 PTS 1

  4. Next go to the Instrument tab and change the thermal profile as follows: 7500 PTS 2

  5. Go to the Auto Increment:7500 PTS 3
Data Analysis
  • Go to ‘View Well Table’ from the Amp Plot screen.
  • Click on the ‘Group By’ button and choose ‘Well Position’ (Column). Then highlight the plate and print the report.

You can also Export Data, and the column format will be retained.

For genotyping data, we recommend TaqMan® Genotyper Software. For relative quantitation, we recommend ExpressionSuite™ Software. (If you have SDS v1.x, you would have to use DataAssist™ software for relative quantitation data.)