How do I add a custom dye to the 7500 or 7500 Fast Real-Time PCR System?

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Answer

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 (approximately 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 (http://tools.lifetechnologies.com/content/sfs/manuals/cms_077749.pdf) for full details.

Answer Id: E7225

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2f351729fe405d2e85c8aac1bbf7f144_FAQ

What is the difference between the ABI Prism® 7000 Sequence Detection System bulb and the Applied Biosystems® 7300, 7500 Real-Time PCR System bulb?

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Answer

The Applied Biosystems® 7300, 7500 and 7500 FAST Real-Time PCR System use the same bulb. It is a halogen lamp that is 12V and 75W with part number 4345287. This is different from the ABI Prism® 7000 bulb which is a tungsten halogen lamp that is 21V and 150W with part number 4347754. You cannot interchange the ABI Prism® 7000 bulb with the 7300/7500/7500 FAST bulb.

Answer Id: E2581

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7fdd335d6b6e9bbd47af43ba6ce1d4f1_FAQ

What are the specifications for the 7500 and 7500 Fast Real-Time PCR Systems?

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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 degrees C
Run Time <2 hr (standard mode)
~35 min (Fast mode - 7500F only)

Temperature Accuracy ±0.25 degrees C (between 35 degrees C and 95 degrees C, after 3 min)
Temperature Uniformity ±0.5 degrees 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

Answer Id: E7223

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db35091ea3c8384caea005c794a34e15_FAQ

Why are there no RQ values for all the samples in my study from the 7500 or 7500 Fast Real-Time PCR System software?

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Answer

The 7500 software requires that the endogenous control be run on every plate within a study. If not, you will only see RQ values for the plate that contains the endogenous control on it. The other plates will have CT values for all the wells, but the software will not calculate RQ values.

If you designed your experiments in this type of layout, you will want to do your analysis in the free DataAssist™ software instead. This program does not have the endogenous control requirement for every plate. Simply export the study results file from the 7500 software as a *.txt or *.csv file, and use that to create a new study in the DataAssist™ software. You should now be able to see RQ values for all samples, even those without an endogenous control on the same plate.

Answer Id: E7241

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bd1c0f2ae8b0eefd9e88ab72c8cf4520_FAQ

If I were to upgrade my existing 7500 System standard unit to the a 7500 Fast configuration, can I still open my old data files?

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Answer

Yes, older 7500 or 7500 fast data files are compatible with the current version of the SDS software. However, there is one exception: please note that the SDS v1.3 software will not allow RQ Studies to be created from RQ Plates from different instrument types, different thermal modes (i.e., fast and standard modes) or different numbers of thermal cycles.

Answer Id: E2341

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e5b95d532b108c510884b99dcdb7d7a0_FAQ

What do I do if the background calibration fails in the 7500 or 7500 Fast Real-Time PCR System?

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Answer

Follow the directions in the 7500 and 7500 Fast Real-Time PCR System Maintenance Guide, Chapter 6 (http://tools.lifetechnologies.com/content/sfs/manuals/cms_077749.pdf), to decontaminate the sample block. You can also watch this helpful video demonstration (https://www.youtube.com/watch?feature=player_embedded&v=dJyolVGhpjk).

Answer Id: E7222

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664bdd6cc64679ce41ad99822d70a4c1_FAQ

What dyes can I use on the 7500 and 7500 Fast Real-Time PCR Systems?

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Answer

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. 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.

Answer Id: E7224

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b5b21017fad8fdb136582ddb8c22c181_FAQ

What volumes can be used in my plate for the 7500 or 7500 Fast Real-Time PCR System?

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Answer

The following volumes are supported for each instrument block:
-7500: 20-100 μL reactions
-7500 Fast: 10-30 μL reactions

Answer Id: E7227

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3b8bfc36b56ec3844132578fd0f1bdae_FAQ

What is the maximum thermal output for the Applied Biosystems® 7500 / 7500 Fast Sequence Detection System in BTU/h?

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The maximum thermal output for the Applied Biosystems® 7500/7500 Fast Sequence Detection System is 3241.5 BTU/h (950W).

Answer Id: E2503

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1437bce7ab843b80f9bef5bbeb348dd0_FAQ

Can I run slower, standard thermal cycling experiments on a 7500 Fast Real-Time PCR System?

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Answer

Yes. you can choose either standard or fast ramp on the 7500 Fast System for software versions 2x or greater, and a 9600 emulation thermal cycling mode is available for customers who wish to run assays based on adjusted ramp rate. However, you should be aware that we cannot guarantee identical performance to the same modes run on a standard 7500 system block.

Answer Id: E2343

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efc57268a95d8b1ba8d6dbe8e51b8ab2_FAQ

How can I run a standard TaqMan® assay reaction on the Applied Biosystems® 7500 Fast Real-Time PCR System?

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Answer

You can run an Applied Biosystems® 7500 Fast Real-Time PCR System instrument in Standard mode as long the following set-up points are followed:

1) The reaction volume is between 10 ul-30 ul
2) The 96-well plate is a MicroAmp® FAST 96 well reaction plate (P/N 4346907, 4346906 or 4366932)
3) Select the Run Mode to be “Standard 7500 “or “9600 Emulation” (2.0.x software versions only offer Standard or Fast - no 9600 emulation choice).

Answer Id: E2474

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2a156d3af7e6a3baeb8dca1e95eff52e_FAQ

What are the thermal cycling parameters of the Fast mode for the 7500 Fast Real-Time PCR System?

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Answer

For the 7500 Fast real time PCR systems, the thermal cycling parameters are defaulted to 50°C/2 min for UNG incubation, 95°C/20 sec for fast polymerase activation, 40 cycles of 95°C/3 secs for the dissociation and 60°C/30 seconds for annealing and extensions.

Answer Id: E2344

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7157c0cb7a6fce420d48265610de1b71_FAQ

What are the ramp rates for the Applied Biosystems® 7500 and 7500 Fast Real-Time PCR Instruments?

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Answer

The sample ramp rates for the 7500 instrument are as follows:

- Standard Mode: 1.6 deg C/sec up and 1.6 deg C/sec down
- 9600 emulation Mode: 0.8 deg C/sec up and 1.6 deg C/sec down. This matches the sample ramp rate achieved for the ABI PRISM® 7700 Sequence Detection System.
- Fast Mode: 3.5 deg C/sec up and 3.5 deg C/sec down.

Answer Id: E2481

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656a6c29f2d9540df35857c505f168d3_FAQ

What is the minimum reaction volume I should be using in the ABI Prism® 7300 and 7500 Sequence Detection System?

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Answer

The minimum reaction volume supported by Applied Biosystems® is 20 ul for Real-Time PCR runs and Allelic Discrimination plate reads on the ABI Prism® 7300 and 7500 (Standard) Sequence Detection System. We have not validated the instruments with reactions volumes below 20 ul. We recommend you perform your own validation for comparable efficiency at lower reaction volume. On the Applied Biosystems® 7500 Fast Real-Time PCR System instrument, you can run a minimum reaction volume of 10 ul reactions.

Answer Id: E2587

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ab8a9d0e0e80c39e4e1605a0734547e4_FAQ

Can I downgrade my Applied Biosystems® 7500 Fast Real-Time PCR System to the standard block?

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Answer

No, we do not offer the option to downgrade an Applied Biosystems® Fast 7500 System to a standard block.

Answer Id: E2315

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47519733cee622ed08673d5ba89e4879_FAQ