Technical FAQs

General Information

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Here you will find an impressive collection of Frequently Asked Questions, organized by subject matter. We hope you will find the information collected here useful. As always, feel free to contact Cepheid Technical Support for any questions or issues not included here.

Support FAQ

Q: How do I contact Technical Support?

Region Telephone Email
US + 1 888 838 3222 Online Support
Australia and
New Zealand
+ 1800 107 884
+ 0800 001 028
Online Support
China + 86 021 5406 5387 Online Support
France + 33 563 825 319 Online Support
Germany + 49 69 710 480 480 Online Support
United Kingdom + 44 3303 332 533 Online Support
Italy + 39 800 902 567 Online Support
South Africa + 27 861 22 76 35 Online Support
Other European, Middle East and
African countries
+ 33 563 825 319
+ 971 4 253 3218
Online Support
Japan + 0120 95 4886 Online Support
Other countries not listed + 1 408 400 8495 Online Support

 

Q: What information do I need to provide when contacting technical support?

A: Please provide:

  • Name
  • Institution
  • Contact Information
  • Instrument Serial Number

Q: Where do I find the serial number?

A: The serial number can be found in several places. These include the back of the instrument where the power cord is attached and at the top of the run report.

RT-PCR FAQ

Q: What are the required materials to perform Real-Time PCR?

A: SmartCycler® & software, computer configured for SC, PCR tubes, primers, probe or intercalating dye, dNTP's, MgCl2, DNA or cDNA, polymerase, water, centrifuge, pipet and pipet tips, buffers. SmartMix and OmniMix are available for convenience which contain all the reagents to perform a PCR reaction, except the primers and probes, in a SmartBead format.

Q: How do I design primers and probes?

A: Information regarding primer and probe design may be found in SmartNote 6.1 Designing Real-Time Assays on the SmartCycler®II System.

Q: How do I optimize my PCR reaction for Real-Time PCR on the SmartCycler®System?

A: Information regarding the design of Real-Time PCR assays may be found in SmartNote 6.1 Designing Real-Time Assays on the SmartCycler®II System.

Information regarding optimizing and analyzing Real-Time Assays on the SmartCycler® System may be found in SmartNote 6.2 Optimizing and Analyzing Real-Time Assays on the SmartCycler® II System.

Q: Which fluorophores should I label my probes with?

A: Information regarding fluorophores may be found in SmartNote 6.3 Dye-Quencher Considerations for the SmartCycler® II System.

Q: Can I run the product on a gel?

A: Yes, you can run the product on a gel by simply using a gel loading pipette tip to remove the contents from the SmartCycler® reaction tube.

Q: Why are there bands of larger DNA than the PCR product on my gels?

A: Possible cancatamers, which is when DNA self anneal to one another and start extending.

Q: Is it possible to perform a multiplex assay in the SmartCycler® II system?

A: Yes, the SmartCycler® II System contains the capability to detect up to 4 targets within the same tube. More targets may be detectable by using the Advance to Next Stage feature in the software. Please refer to SmartNote Advance to Next Stage.

Q: I have a positive growth curve in my negative control when I use an intercalating dye. What is this?

A: This may be a result of non-specific amplification. Please refer to SmartNote 6.1 Designing Real-Time Assays on the SmartCycler® II System.

This may be a result of primer-dimer generation. To minimize primer-dimer formation, please follow these guidelines:

  • Use a hot-start enzyme to minimize extension of non-specific binding of primers during the reaction preparation.
  • Determine the minimum amount of time for each step in the thermal cycling protocol and avoid excessive hold times.
  • Use the lowest concentration of primer necessary for efficient amplification.
  • Use the highest annealing temperature possible.
  • Use the minimum number of cycles.
  • Use the lowest concentration of MgCl2 concentration.
  • Design primers with the highest Tm possible.
  • Design primers that do not contain homologous sequences.
  • Avoid C's and G's at the 3'end of the primers.

Protocols FAQ

Q: When I use a protocol that contains a 30∞C hold, the processing block turns off a few minutes after the run is started.

A: The acceptable temperature range is 40∞C-98∞C. If the specified temperature is not reached in 5 minutes the site will automatically stop. The lowest temperature in the protocol must be above the internal temperature of the instrument.

Q: Can I add more cycle repeats while an experiment is in progress?

A: Although the software does not allow for the addition of cycle repeats while a run is progress, it is possible to program an excess of cycle repeats and stop the run or sites when appropriate.

Q: Can I read optics during multiple steps in a single stage?

A: No, only one optical read per stage can be programmed.

Q: Can I use the SmartCycler® II System as a tradition PCR instrument?

A: Yes, you can use the SmartCycler® II System as a traditional thermocycler by turning the optics OFF during the cycling protocol.

Q: Can I perform a melt from a high temperature to a low temperature?

A: Yes. The lowest temperature must be greater than internal temperature of the instrument.

Q: How many first derivative peaks are displayed on the melt graph?

A: Multiple first derivative peaks indicate that multiple products were amplified during the PCR. Generally, primer-dimers melt before specific products. To change the number of melt peaks that are displayed on the graph or to change the qualifying height for the first derivative peaks, click Setup menu>System Defaults>Melt Settings. If the melt curve appears noisy (jagged), try smoothing the melt curve by averaging 3-5 points. Click the Setup menu>System Defaults>Melt Settings.

Graphs & Optical FAQ

Q: What dyes can be used on the SmartCycler® II System?

A: The SmartCycler® II System is calibrated for FAM, Cy3 or TET or Alexafluor532, Texas Red and Cy5 or Alexafluor647.

Q: Can I use VIC, HEX or JOE on the SmartCycler®?

A: VIC, HEX, and JOE are very similar to TET and therefore may work with our calibration, but we cannot guarantee their performance since the base it is attached to may shift the optical spectrum. To use a dye that has not been calibrated at Cepheid, we suggest running as a simplex reaction and viewing the fluorescence reading in the adjacent channels to determine any cross-talk. If the desired result is not generated, the SmartCycler® and SmartCycler® II system may be recalibrated with the user-defined Optical Calibration feature in the SmartCycler® Software.

Q: I created a FAM graph but it is displaying temperature.

A: This is most likely due to the incorrect graph type. Check the graph definition in the Define Graphs screen to verify that the graph type is optics and not set to temperature.

Q: I created a new graph but it is not listed in the View Results screen.

A: When a new graph is defined in the Define Graphs screen it is saved and stored in the database but not associated with a run unless the "Automatically add to new runs" box is checked. To view a new graph, go to the View Results screen and click the Select Graph button at the bottom of the screen. Highlight the graph name in the "All Graphs" column, click the right-pointing arrow then click OK.

Q: When I use the print icon, the graph print out does not contain enough information, is there an alternative way to capture a picture of the data?

A: To capture the entire screen use the Print screen button on your keyboard. Simultaneously hold down the Print Screen and Alt buttons on the keyboard then open Word or Power Point and select paste.

Also, it is possible to save the screen as a jpeg file and to save graphs as jpeg files. To save the screen as a jpeg file go to the Tools menu and select Save Screen to File. To save a graph as a jpeg file, right-click on the graph and select Save to file (jpg).

Q: I can't zoom on a graph when a run is in progress.

A: Since data is constantly being collected and reported in real time, it is not possible to hold a zoom from a previous time point. This occurs even when viewing a saved run if another run is in progress.

Q: How can I get negative fluorescence?

A: This is caused by the background subtraction calculation. This usually occurs when the threshold is set too high. If the primary signal or 2nd derivative does not cross the threshold, the background subtraction calculation will continue until the end of the run. Try decreasing the threshold value or adjusting the background min and max values.

Q: Do I need to select graphs before I start a run?

A: It is not necessary to define or select graphs before the run is started. Graphs can be created before, during, or after the run is started.

Q: Can I look at other runs while the instrument is in use?

A: Yes, it is possible to view and analyze previous runs while the instrument is in use.

Analysis FAQ

Q: What is a Ct?

A: The most basic definition of a Ct is the first cycle in which there is a significant increase in fluorescence above the background.

Q: What is the difference between 2nd derivative and primary curve?

A: The 2nd derivative and primary curve are two ways to determine a cycle threshold value.

If the primary curve is used the cycle threshold reported is the intersection of the growth curve and the threshold.

If the 2nd derivative is selected the software determines the cycle threshold value by calculating the 2nd derivative of the growth curve. The software chooses the largest 2nd derivative peak for the cycle threshold (this is usually the inflection point of the growth curve).

Q: What is the difference between automatic and manual Threshold Settings?

A: If manual is selected, the user sets the fluorescence value used for the threshold. Manual mode gives the user more flexibility to evaluate background.

Automatic will always set threshold above background, however, it does not allow the user to choose the fluorescence value used for the threshold. Automatic mode calculates the threshold as the specified number of SDs above the background.

Q: I made changes to the analysis table but the graph did not update to reflect changes.

A: The Update Analysis button located at the bottom of the View Results screen must be selected to implement changes made to the Analysis Settings or Results Table.

Q: Is there a way to reduce the noise in my growth curves?

A: The boxcar average analysis setting averages data points, which reduces the noise displayed in the growth curve. The number of points (usually 2 or 3) is specified by the user. However, boxcar averaging can shift cycle threshold values by approximately one cycle.

Q: When background subtraction is "ON" I see drift of the baseline (positive or negative). Can this be corrected?

A: The background min and max cycle numbers can be adjusted to reduce the baseline drift.

Q: My negative samples are reporting Ct values in the Results Table.

A: If the threshold is set too low and baseline drift crosses the threshold a Ct may be reported. Increase the threshold value (manual mode) or the number of SDs used to calculate the automatic threshold.

Q: Can I make changes to the Analysis or Results table while a run is in progress?

A: Yes, changes can be made to the analysis settings at any time. Always click the Update Analysis button to implement changes made to the Results Table or Analysis Settings.

Note: If the Advance to Next Stage feature is used in software version 2.0, the analysis settings must be set up before the run has started.

Q: Can I use one standard curve for several runs?

A: Yes, a previous standard curve can be imported and saved with a run that contains only unknown samples. Click the Import Std Curve button at the bottom of the View Results screen to select a run from a list of all the valid standard curves in the database. (Chapter 5)

Q: Do I need to set-up the analysis settings before a run is started?

A: No, the analysis settings can be set up before, during or after a run. Each time a change is made to the analysis settings the Update Analysis button must be selected to implement the changes. To save changes made to the Analysis settings click the Save Run button.

Q: I selected sites as standards but my standard curve graph does not show any data.

A: The Update Analysis button must be selected after changes are made to the Results Table. Check that the standard values were entered in the correct column.

Reports & Data FAQ

Q: Where is the exported data saved?

A: Exported data is saved to the export folder that is located in the C:Drive>SmartCycler® >Export (creating a shortcut to this folder is recommended).

Q: How do I see errors that have occurred?

A: Notification of an error is displayed in the Check Status screen and the Results Table. To view errors that have occurred since software application was last opened go to the messages field in the Maintenance screen.

To view errors that occurred before software was last opened go to the Errors folder that is located in the C: drive>SmartCycler®>Errors folder. It is necessary to open notepad to view error messages. These will also be posted on the last page of the report.

Q: How do I export? What can I do with the exported data?

A: Select the Export button at the bottom of the View Results screen. Select the type of data to export. Enter a file name and click Save.

Maintenance FAQ

Q: What is the GeneXpert® System?

A: The GeneXpert Dx System automates and integrates sample preparation, nucleic acid amplification, and detection of the target sequence in simple or complex samples using real-time Polymerase Chain Reaction (PCR). The system is suited for in vitro diagnostic and research based applications that require hands-off processing of patient samples (specimens) and provides both summarized and detailed test results data in tabular and graphic formats.

The GeneXpert system can only be used with the GeneXpert cartridge.

Q: What is the cartridge?

A: Disposable, single—use GeneXpert Dx cartridge holds the samples and reagents that you want to process in the GeneXpert Dx System. Each cartridge consists of the following components:

Processing chambers—Hold samples, reagents, processed sample, and waste solutions. One chamber is designated as an air chamber to equilibrate pressures within the cartridge.

Valve body-Rotates and allows fluid to move to different cartridge chambers and to the reaction tube. Within the valve body, the specimen is isolated, PCR inhibitors are removed, and specimens are ultrasonically lysed (if applicable). After the sample is processed, it is mixed with PCR reagents and moved into the integrated reaction tube.

Reaction tube—Enables rapid thermal cycling and optical excitation and detection of the tube contents. The reaction tube is automatically inserted into the I-CORE module after the cartridge is loaded into the instrument.

The GeneXpert cartridges are not supplied with the Cepheid. Contact Cepheid Customer Service to order the assay specific cartridges: 1-888-838-3222 Option 1.

Q: What dyes can be used on the GeneXpert DX system?

A: The GeneXpert (Diagnostic System) is calibrated with FAM, Alexa Fluor532, Texas Red, and Alexa Fluor647.

Q: What dyes can be used on the GeneXpert Biothreat system?

A: The GeneXpert (Biothreat System) is calibrated with FAM, VIC, DRox and LIZ

Q: What assays can I run on the GeneXpert DX System?

A: The GeneXpert® Dx system can run a number of assays developed by Cepheid. Please refer to the products section of our website.

Q: What symbology is supported with the 2D scanner?

A: Codes 39 and 128 are supported.

Q: Can I connect a different scanner or change the configuration?

A: No, we can only support the components that are shipped with the system. Do not replace or reconfigure any of the components. Using non-approved components can produce invalid results, cause loss of data, or damage other system components.

Q: How do I archive a test?

A: Click on Data Management/ Archive Test and click on the test(s) you wish to archive. Click Archive and follow the defaults. When emailing an archive run, do not use a Yahoo account or a MAC computer. Also, some filters may remove the .gxx extension. If this happens, change it to anything but .txt.

Q: How many GX instruments can be hooked up to a computer system?

A: The computer can support 1 GX instrument.