How do I add a custom dye to the 7500 or 7500 Fast Real-Time PCR System?
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: 7225
What is the difference between the ABI Prism® 7000 Sequence Detection System bulb and the Applied Biosystems® 7300, 7500 Real-Time PCR System bulb?
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: 2581
What are the specifications for the 7500 and 7500 Fast Real-Time PCR Systems?
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: 7223
Why are there no RQ values for all the samples in my study from the 7500 or 7500 Fast Real-Time PCR System software?
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: 7241
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?
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: 2341
What do I do if the background calibration fails in the 7500 or 7500 Fast Real-Time PCR System?
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: 7222
What dyes can I use on the 7500 and 7500 Fast Real-Time PCR Systems?
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: 7224
What volumes can be used in my plate for the 7500 or 7500 Fast Real-Time PCR System?
Can I run slower, standard thermal cycling experiments on a 7500 Fast Real-Time PCR System?
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: 2343
What is the maximum thermal output for the Applied Biosystems® 7500 / 7500 Fast Sequence Detection System in BTU/h?
How can I run a standard TaqMan® assay reaction on the Applied Biosystems® 7500 Fast Real-Time PCR System?
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: 2474
What are the thermal cycling parameters of the Fast mode for the 7500 Fast Real-Time PCR System?
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: 2344
What are the ramp rates for the Applied Biosystems® 7500 and 7500 Fast Real-Time PCR Instruments?
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: 2481
What is the minimum reaction volume I should be using in the ABI Prism® 7300 and 7500 Sequence Detection System?
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: 2587
Can I downgrade my Applied Biosystems® 7500 Fast Real-Time PCR System to the standard block?