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Applied Biosystems™ StepOne™ Real-Time PCR System Spectral Calibration Kit (48 well)

Catalog No. 4371433
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Catalog No. 4371433 Supplier Applied Biosystems™ Supplier No. 4371433
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Description

Includes

Contains three 48-well reaction plates - one background plate and two containing dye standards(plates I & II). Plate I contains FAM, JOE and ROX; Plate II contains SYBR™ Green I and VIC.

Easy-to-use kit establishes the pure dye spectra and multicomponent values for FAM™/SYBR™ Green I, VIC™/JOE™, and ROX™ dyes on the StepOne™ Real-Time PCR System.

The Applied Biosystems™ StepOne™ Real-Time PCR System Spectral Calibration Kit contains one background plate and three preloaded, 48-well fast optical reaction plates containing five dye standards.

  • Easy-to-use
  • Provides accurate discrimination of the fluorescence signal of individual dyes in the reaction mixture
  • Includes the Passive Reference I dye standard for signal normalization in all real-time and endpoint reactions

Order Info

Dry ice shipping; store at -15° to 25°C; protect from light; expiration date: 12 months from the date of delivery.

Specifications

Content And Storage Contains three 48-well reaction plates—one background plate and two containing dye standards(plates I & II). Plate I contains FAM, JOE and ROX; Plate II contains SYBR™ Green I and VIC.

Store at -15 to 25°C. Protect from light.

Expiration date: 12 months from the date of delivery
Calibrated Dye FAM, JOE, ROX, VIC, SYBR™ Green
For Use With (Equipment) StepOne™, Fast Mode, StepOne™, Standard Mode
Detection Method Fluorescence
Product Line StepOne
Product Type Spectral Calibration Kit
For Use With (Application) Real Time PCR (qPCR)
Shipping Condition Dry Ice
Quantity 1 kit
Calibration Type Spectral Calibration

Frequently Asked Questions (FAQs)

What does "Error - Cannot calculate Pure Dye Matrix" mean?

This issue can occur if the run is interrupted. To prevent this from happening in the future, avoid ending the PCR with an infinite hold stage or interrupting the run. Please send the file to techsupport@thermofisher.com so that our software group can attempt to recover the data.

What does "Error - Fatal Error Number 0x8158" mean?

This is generally caused by incorrect placement of tube strips, resulting in a sample block that can't properly be lowered. We recommend contacting Instrument Services to have an engineer walk you through a manual drawer-lowering process during instrument power-up. To prevent this from happening in the future, place tubes toward the middle of the block or use a placeholder strip on the opposing side to counterbalance block pressure.

What does "Error - Java.lang.IllegalArgumentException: One of the raw spectra is null" mean?

This issue can occur if the “Start Run” button is clicked more than once or if illegal characters are used in the experiment/file name. You can recover the data yourself by overriding the calibration file with one that represents the calibration status of the instrument when you ran that particular file. Use the toolbar at the top to navigate: Analysis then Override Calibration then Use Calibration From Another File.

What does "Error - Cannot open the file because file contents are not recognized" mean?

This issue can occur if duplicate samples are added to the experiment, if illegal characters are used in the experiment/file name, or if the file extension was changed to .eds from a different extension.
Please send the file to techsupport@thermofisher.com to attempt recovery.

What does "Error - Analysis failed due to GExSession doesn't exist in context" mean?

This error means that the data collection was not turned on as appropriate. If this is inadvertently turned off, no data will be collected. Thus, the data are not retrievable in such a file.

What does "Error - Analysis failed due to: 1" mean on the StepOne or StepOnePlus Real-Time PCR System?

Please send the file to techsupport@thermofisher.com so that we can have our software group attempt to recover the data.

What should I do when I get the error "Error - Cannot connect to the instrument" on the StepOne or StepOnePlus Real-Time PCR System?

Check that all the cables are correctly plugged into the respective ports and not bent. Follow the directions on page 135 of the StepOne and StepOnePlus Maintenance Guide (https://tools.thermofisher.com/content/sfs/manuals/4376782.pdf) to check the connectivity. If the issue is still not resolved, please contact Instrument Services at 1-800-955-6288, option 3, option 1, or by email at instrumentservices@thermofisher.com for further assistance.

What does "Error - Failure to acquire needed controlling rights" mean?

This error usually occurs when you first start your run. Reboot the instrument and restart the computer. Make sure to log into the computer as the Administrator (on the Windows OS).

Where are my files saved to on the StepOne and StepOnePlus Real-Time PCR Systems?

Run files will be saved to both the instrument and the connected computer. On the connected computer, files will be saved to the default data folder, unless you change it.

To find or change the default folder, go to Tools then Preferences then 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.

How do I set up a touchdown PCR on the StepOne and StepOnePlus Real-Time PCR Systems?

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.

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.

What plates or tubes can I use with the StepOne and StepOnePlus Real-Time PCR Systems?

Please refer to this selection table (https://www.thermofisher.com/us/en/home/life-science/pcr/pcr-plastics/real-time-pcr-plastics.html) for compatible plates, tubes, and films.

How do I load tube strips on the StepOne and StepOnePlus Real-Time PCR Systems?

Follow these instructions for proper loading of tubes, tube strips, and plates:

- Put the tubes or tube strips in the tray with the adapter on the 96-well support base.
- If using a plate, put the plate directly on the 96-well support base.
- Pipet your reactions into the tubes/tube strips or the wells on the plate.
- Seal the tube/tube strips with optical flat caps.
- If using a plate, seal the plate with either optical adhesive film or flat caps.

For optimal performance on the StepOnePlus instrument with partial loads:
- Load at least 16 tubes and arrange them in adjacent columns of 8 tubes, using rows A through H, or adjacent rows of 8 tubes, using columns 3 through 10.
For optimal performance on the StepOne instrument, load at least 4 tubes in the sample block.

What volumes can be used in my plate for the StepOne or StepOnePlus Real-Time PCR Systems?

The StepOne and StepOnePlus Real-Time PCR Systems have a Fast block, which supports reaction volumes of 10-30 µL.

Can I reuse my calibration plates for the StepOne or StepOnePlus Real-Time PCR Systems?

The calibration plates can be stored and reused three times for up to one year after you receive them, so make sure to return them to their original packaging and return them to -20 degrees C storage until the next use. If needed, you can make your own background plate using deionized water. Please follow the directions in the StepOne and StepOnePlus Maintenance and Administration Guide (Appendix C) (http://tools.thermofisher.com/content/sfs/manuals/4376782G.pdf) for more details.

When do I need to run the RNaseP calibration for the StepOne or StepOnePlus Real-Time PCR Systems?

The RNaseP verification plate is a single-use plate that 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 that 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. It is also recommended to use after the instrument has been moved to a new location. For the StepOne instrument, the RNaseP plate is Cat. No. 4371439. For the StepOnePlus instrument, the RNaseP plate is Cat. No. 4351979.

When do I need to calibrate the StepOne or StepOnePlus Real-Time PCR System?

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 18 months
Spatial calibration: Every 18 months
RNaseP instrument verification: After the instrument has been moved, or as needed to verify instrument performance

What type of Real-Time PCR (Sequence Detection System) information is available from Applied Biosystems?

Tutorials demonstrating introductions to Real-Time PCR, guidelines for designing real-time assays using Primer Express software, and various instrument set-ups can be found under Life Technologies University on our website. User bulletins, manuals and Product inserts, protocols can be found as downloadable pdf files on our Technical Resources page.

For Research Use Only. Not for use in diagnostic procedures.