What are the extensions of files generated by the ABI PRISM® 7000 Sequence Detection System, Applied Biosystems® 7300, 7500, 7500Fast, 7900HT Fast Real-Time PCR System, StepOnePlus™ Real-Time PCR System, and ViiA™ 7 Real-Time PCR Systems?

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All run files have an .SDS or .EDS extension. A Relative Quantification study will have an .SDM or .EDM extension and a template will have an .SDT or .EDT extension. The software for StepOnePlus™, ViiA™ 7, and 7500 2.0.x software have .EDS, .EDT, or .EDM extensions; all other software have .SDS, .SDT, or .SDM extensions.

Answer Id: E2473

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b338dcad4543321f7a5058c6a1189fcd_FAQ

What computer configurations can I use with the ViiA™ 7 Fast Real-Time PCR System?

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The current recommended configuration is the Dell Gx980 tower computer running Windows® XP SP3. Note that the instrument does not require a computer to start a run. Any other computer configurations are not fully tested.

Answer Id: E2328

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a85e6c43593177fa4c71ed35083de301_FAQ

Can I use a custom dye on my Applied Biosystems® 7500, 7500 Fast, 7900HT Fast or other Real-Time PCR System?

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Yes, you can run custom dyes, but the custom dye must fall into the wavelengths that are capable of being detected by a specific instrument. For optimal results on the filter-based instruments (including 7500 and 7500 Fast), the custom dye emission wavelength must fall into one of the emission filter peak detection wavelengths. For the laser-based 7900HT systems, the custom dye wavelength must fall within the overall range of detection for the instrument. Please refer to the Real-Time PCR information pages on our website under “Products & Services” for profiles of each instrument with a list of compatible standard dyes and the corresponding wavelengths supported. Please also refer to your User Manual or Installation and Maintenance Manual for instructions on how to calibrate custom dyes on your particular instrument.

Answer Id: E2457

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0ca47dadf0d94c86e854a69c5813149b_FAQ

What are some of the main differences between Applied Biosystems® Real-Time PCR Systems, like the 7500, 7900HT, 7700 or 7000 Sequence Detection System instruments?

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The systems most often vary by the light source and filter sets. The 7000, 7300, 7500, and 7500 Fast systems utilize a halogen lamp as the excitation source and emission filters to detect the emission fluorescence. The 7000 and 7300 systems both have one excitation and four emission filters, while the 7500 and 7500 Fast systems have five excitation and five emission filters. Five excitation and five emission filters allow the 7500 and 7500 Fast systems to detect dyes in the far red range. The 7700 and 7900HT systems utilize an argon ion laser as the excitation source and the instrument scans across all wavelengths between 520 nm to 610 nm.

The 7500, 7500 Fast and 7900HT systems also offers the ability to upgrade to the Fast chemistry; typical real-time runs take about 30-45 minutes.

In addition, the 7900HT system offers further high throughput options such as a 384-well block and a TaqMan® Low Density Array Upgrade as well as a Zymark Twister Automation Accessory that will robotically load plates on the instrument.

For further information please speak with your local Life Technologies sales representative.

Answer Id: E2459

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1b49dd627a1d48194fcd174fd632c10b_FAQ

Can any of the Applied Biosystems® Real-Time PCR Systems detect one copy?

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All Applied Biosystems® instrument systems are qualified to detect a two-fold change. Scientifically speaking, there is a statistical calculation that predicts the probability of detecting one molecule in any given sample called the Poisson Distribution. The Poisson Distribution predicts that one molecule will be detected 66% of the time. This is assuming that the assay has been optimized to the fullest capacity and that there are no aliquoting or pipetting errors involved.

Please refer to the following paper concerning one-copy detection. Lockey, C., E. Otto, and Z. Long. "Real-time fluorescence detection of a single DNA molecule." BioTechniques 24 (1998): 744-746.

Each assay designed on one of our platforms needs to be optimized to determine the Linear Dynamic Range of the assay. An Applied Biosystems® Sequence Detection System (Real-Time PCR instrument) may be able to detect one copy in a fully optimized assay. As stated above, one copy can only be detected 66% of the time in a fully optimized system.

Answer Id: E1824

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811ac584aae5c0af52f97013a84eabdc_FAQ

When do I need to calibrate the ViiA™ 7 Real-Time PCR System?

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Answer

The recommended maintenance schedule and calibration instructions can be found in the User Guide here: http://tools.lifetechnologies.com/content/sfs/manuals/4442661C.pdf
In brief, the background calibration is recommended to be done every month. All other calibrations (pure dye, ROI, uniformity, and normalization) are recommended to be done every six months.

Answer Id: E7183

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a89654c2b2ee0245f095c546cf9fbddb_FAQ

What are the uniformity and normalization calibrations for on the ViiA™ 7 Real-Time PCR System?

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Answer

The uniformity calibration generates data that allow the software to compensate for the physical effects of the ViiA™ 7 system filters using the ROI plate. The normalization calibration generates factors that the software uses when comparing data from multiple ViiA™ 7 instruments within a study. Two normalization plates are provided in the calibration kit, one containing FAM™/ROX™ dyes and the other with VIC®/ROX™ dyes.

Answer Id: E7184

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791370dbdc977653643ee676fd27808f_FAQ

Can I reuse my calibration plates?

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The calibration plates can be stored and reused three times for up to 1 year after you first open them, so make sure to return them to their original packaging and return them to -20 degrees 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. Please follow the directions in the ViiA™ 7 Real-Time PCR System Installation, Networking, and Maintenance Guide for more details at http://tools.lifetechnologies.com/content/sfs/manuals/4442661C.pdf.

Answer Id: E7185

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031b48cfb280d589b98ef969b72fb0ac_FAQ

Can I create my own calibration plates?

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Answer

You can create your own background calibration plate using the directions in the ViiA™ 7 Real-Time PCR System Maintenance Guide (Appendix C) http://tools.lifetechnologies.com/content/sfs/manuals/4442661C.pdf. For all other calibration plates, you can purchase a calibration kit, or the individual plates, as required (http://www.lifetechnologies.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-reagents/real-time-calibration-kits/viia-7-system-calibration-kits.html).

Answer Id: E7187

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d62f6cd29f7af3b5141aa0f7e8f701c1_FAQ

How do I set up a touchdown PCR experiment on the ViiA™ 7 Real-Time PCR System?

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Answer

In a touchdown PCR experiment, you will change either 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 higher in the early cycles and the efficiency in the later cycles. In this example, we will set the method to do the following:

1. Go to File then New Experiment then Advanced Setup. Fill out the relevant options as you normally would.
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 and click ‘Save Setting’. You will then see a green triangle show up next to the parameter you changed. Your new method has now been applied.

Answer Id: E7188

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f98938edb80fb5c55f917b4928a3a770_FAQ

How do I add a custom dye to the ViiA™ 7 Real-Time PCR System?

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You will need an oligo sequence with the custom dye but without the quencher molecule. For each custom dye, prepare samples in a concentration range of 100-2,000 nM, choosing a 2-3-fold difference in dilution points. Use either the background buffer provided in the calibration kit or your own buffer to dilute the oligo. A full plate is not needed; see the example below.

1. Set up a dummy run using the ‘Standard Curve’ option. Alter the thermal profile so that it simply ramps to 60 degrees C with a 2 min hold. Ensure that the filters of interest are selected.
2. When the run is complete, export and examine the raw data. Select the concentration to use by finding the dilution that will give you an acceptable signal in the following ranges:
- For a 384-well plate: between 400,000 and 1,200,000
3.For a 96-well plate: between 1,400,000 and 4,300,000
4.Create a full plate of the dye using the selected concentration and run the custom dye calibration as normal, using 20 μL. (Refer to the ViiA™ 7 Real-Time PCR System Maintenance Guide, “Creating a custom dye plate”, for full details.)

Answer Id: E7189

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7e5119ed3331fce7f681829509d4e2cb_FAQ

How many files can the ViiA™ 7 Real-Time PCR System hold?

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The ViiA™ 7 Real-Time PCR System can store up to 100 runs on the instrument itself.

Answer Id: E7190

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298d40acfd55d8cec6f2ed2022df0f35_FAQ

What volumes can be used in my plate on the ViiA™ 7 Real-Time PCR System?

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Answer

The following volumes are supported for each instrument block:

- 96-well standard (10-100 μL reactions)
- 96-well Fast (10-30 μL reactions)
- 384-well (5-20 μL reactions)
- TaqMan® Array Micro Fluidic Cards (approximately 1 μL reactions)

Answer Id: E7191

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f8645d2cd09ad25081386552ba21b4d1_FAQ

Can I collect data at two separate steps for analysis in the ViiA™ 7 Real-Time PCR System?

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Answer

Yes. The ViiA™ 7 system can collect data at multiple steps in the amplification stage. You will need to turn on the data collection for every step of interest. In the analysis, only one set of data can be displayed at a time. To change the data set used for analysis, from your data file go to Analysis Settings then Ct Settings and change the dropdown under ‘Data Step Selection’.

Answer Id: E7192

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ca4682eba76b12cf4be72a6010db9918_FAQ

What can I use for data analysis on the ViiA™ 7 Real-Time PCR System?

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Answer

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

Answer Id: E7193

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1b4b50d9c99e715046999362a7b02b0f_FAQ