I got an error message on the Applied Biosystems® 7900HT Fast Real-Time PCR System that says to check the log file. What is this and where do I find it?
The log file tracks events during the course of an instrument run. To locate the log file, open up the hard drive of the instrument computer. Open the Applied Biosystems® folder, then the SDS 2.x.x folder, then the log folder. The log file is a Microsoft® Word file in .doc format and is saved as the date the run was initiated.
Answer Id: 2460
How can I send .sds and .sdm data files to the Real Time PCR Technical Support team?
These files are often large (>5 MB) and do not always transfer well through standard email. Please visit Contact Us on our website and reach out to your local Technical Support team first. They can then advise you on other methods we can use to share large files with you, such as .ftp or similar programs. Please be prepared to provide us with a detailed summary of your experiment, including information such as your instrument model number, assay ID, template input, master mix used, and thermal profile.
Answer Id: 2461
Can I use probes with a Black Hole Quencher (BHQ) on Applied Biosystems® Real-Time PCR Systems?
Yes, you can use probes with a BHQ on Applied Biosystems® Real-Time PCR Systems. However, you will need to set the quencher to NONE. Here is an example of a quencher setting based on the SDS software for the Applied Biosystems® 7500 Real-Time PCR System:
(1) After you create a new file, please click on TOOLS in the menu bar and choose DETECTOR MANAGER.
(2) Click FILE button on the lower left corner of the DETECTOR MANAGER window and choose NEW.
(3) In the NEW DETECTOR window, choose (NONE) in the dropdown list of QUENCHER DYE.
Answer Id: 2462
What is the concentration of primers in the primer-limited TaqMan® Endogenous Control Assays and what function does it have in my reaction?
The final primer concentration for the primer-limited Endogenous Controls is 150 nM for the forward primer and 150 nM the reverse primer. Usually, the endogenous control genes are abundantly expressed. The limited primer concentration will force the PCR of the endogenous control to plateau early, so that it leaves enough dNTPs and enzyme for lower expressed target genes to be amplified.
Answer Id: 2463
Can I check the end product of a TaqMan® Gene Expression Assay by running an agarose gel?
Yes, you can check the end product on an agarose gel. If you use the TaqMan® Universal PCR Master Mix (including AmpErase® UNG, Cat. No. 4304437), we recommend running the gel immediately after the PCR, since the trace amount of AmpErase® UNG may digest the end product that contains double-stranded DNA with dUTP incorporated. The average length of amplicon ranges from 50-150 bp. The exact amplicon length is indicated on the detailed page of each assay on our website. You can get to that page by clicking on the Assay ID; the amplicon information is listed in the section "Assay Details".
Answer Id: 2464
Should I run a pure dye calibration each time I exchange the sample block of the Applied Biosystems® 7900HT Fast Real-Time PCR System?
You have to run a calibration when a new sample block format is used in the 7900HT Fast Real-Time PCR System, but it is not necessary to run the pure dye calibration if the block has been used before. For example, if you use a 384-well sample block for the first time, then you have to run a 384-well block calibration. Later, should you switch to a 96-well block and then switch back to a 384-well block again, then there is no need to rerun the calibration of the 384-well sample block. We recommend that you run a background run each time you exchange the sample block because dust particles or other contaminants could get into the wells while the block is being stored outside of the instrument. Please consult the 7900HT User Manual, Cat. No. 4317596, for the recommended maintenance procedures.
Answer Id: 2465
I received an email saying that the Custom TaqMan® Gene Expression Assay I ordered failed manufacturing. What does this mean and what can be done?
After the primers and probe are designed for a Custom TaqMan® Gene Expression Assay, they will be sent for synthesis. Certain primer and probe sequences cannot be synthesized to pass our strict Quality Control procedure. We will try one more time to synthesize the oligos. If the second synthesis fails an email will be sent, notifying you that the order failed manufacturing. You will not be billed if the assay is not manufactured.
If your assay fails during manufacturing you will need to design a new set of primers/probe and send the oligo sequences to us for custom oligo synthesis. If you have an Applied Biosystems® real-time PCR instrument, you should have a copy of Primer Express™ software, which can be used for general primer/probe design.
Answer Id: 2466
When I start the run with 7300, 7500, and 7500 Fast Real-Time PCR Systems, I get an error message that states Fatal error, block upswitch not engaged. How can I correct this problem?
The error message is caused by a shifting of the heated cover. You can correct the problem by following this procedure:
(1) Shut down both the software and the instrument.
(2) Open the front cover by inserting and applying force with a pipette tip or a small Allen wrench on the right side of the front cover.
(3) Inside of the machine, you will see a metal plate with a plastic handle. This metal plate attaches to the heated cover. Pull the handle toward the front of the instrument and then close the front cover.
To prevent this from happening again, make sure you push the PCR plate completely down into the precision plate holder. Also, do not use compression pads on 7300, 7500, and 7500 Fast Real-Time PCR Systems.
Answer Id: 2468
I lost my user manuals for the 7300 and 7500 Fast Real-Time PCR Systems. How can I get an additional copy?
What dyes can I use as reporter dyes in a multiplex reaction on the Applied Biosystems® 7300 Real-Time PCR Systems?
The Applied Biosystems® 7300 Real-Time PCR System has 4 signal detection filters: Filter A (FAM™ and SYBR® Green), Filter B (VIC® and JOE™), Filter C (TAMRA™, NED™ and Cy3®) and Filter D (ROX™ and Texas Red®). For multiplexing reactions, the reporter dyes from the different assays should be chosen from different filter sets, so they can be compatible. In addition, the reporter dye need to be compatible with the quencher and the passive reference dye that might be present in the reaction. The commonly used combination is FAM™ and VIC®.
Answer Id: 2471
What dyes can be used as a reporter in a multiplex reaction on Applied Biosystems® 7500 Real-Time PCR System?
The following dyes can be used as the reporter dye with MGB-NFQ as the quencher on the Applied Biosystems® 7500 Real-time PCR System or Applied Biosystems® 7500 Fast Real-Time PCR System when using TaqMan Gene Expression Master Mix (recommended for multiplexing) or any other Applied Biosystems® TaqMan® master mix:
FAM™, VIC®, JOE™, NED™, TAMRA™,CY3®, ROX™, TEXAS RED®, CY5® dyes.
Of these dyes, we recommend a duplex reaction with FAM™ and VIC® or FAM™ and NED™ dyes; if you are triplexing, we recommend FAM™, VIC®, and Cy5® or FAM™, VIC®, and TAMRA™ dyes. You must choose dyes that are well separated in emission wavelength. If you are using any probes with a TAMRA™ dye quencher, you cannot use NED™, TAMRA™, or CY3® dye as one of the reporter dyes in that multiplex reaction. ROX™ or TEXAS RED® dyes can be used as reporter dyes only when not using an Applied Biosystems® Master Mix.
Answer Id: 2472
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?
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: 2473
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
Can I move the Applied Biosystems® 7300, 7500, or 7500Fast Real-Time PCR System to another room or another building by myself?
Yes, you can move the instrument carefully on your own. It is recommended that you recalibrate the instrument with the pure dye calibration kit(P/N 4349182, 4349180, 4351151, 4360788, or 4362201) and run an RNase P instrument verification plate (P/N 4351979 or 4350584), after the move.
Answer Id: 2476
How can I export the amplification plot or gene expression plot from the Applied Biosystems® 7300, 7500, and 7500 Fast Real-Time PCR System’s software?
With SDS 1.4 software, right-click on the graph, and choose "Export as JPEG" or "Export to Powerpoint". With SDS 2.0.x (for 7500/7500fast only), you can either click on the save icon above the graph "Save as JPEG Image" or click on the Export drop-down arrow (located on the upper toolbar) and choose "Send to Powerpoint"
Answer Id: 2477