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View additional product information for Custom TaqMan™ Gene Expression Assay, VIC primer-limited - FAQs (4448488, 4448487, 4448492)
36 product FAQs found
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".
To reorder the same Custom TaqMan Assay, you can follow these steps:
- Select an appropriate catalog number, considering the size of the assay that you want. You can find the different catalog numbers for Custom TaqMan Assays on their respective product pages on thermofisher.com.
- Once you have identified the necessary catalog number, go to www.thermofisher.com and click on "Quick Order", in the top right corner of the screen.
- In the following page, paste your selected catalog number in the "Catalog Number" field and paste the assay ID from your previous order in the "Assay ID" field.
- Click "Add to cart" and then in the following screen click "Proceed to check out".
While TaqMan Gene Expression Assays can be used to perform a one-step RT-PCR reaction, these assays have not been optimized for this application. TaqMan Gene Expression Assays have been designed, tested, and optimized using Applied Biosystems universal conditions for a two-step RT-PCR reaction.
When preparing the TaqMan or Custom TaqMan Gene Expression Assays, the recommended and supported volume is 20-50 µL for a 96-well standard plate set-up; 10-20 µL for a 96-well fast plate set-up and 5-10 µL for a 384-well set-up. For more information, please consult the TaqMan Gene Expression Assays and Custom TaqMan Gene Expression Assays protocols.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
For optimal performance of the TaqMan and Custom TaqMan Gene Expression Assays, use 1 to 100 ng of cDNA per 50 µL total reaction volume. For more information, please consult the TaqMan Gene Expression Assay protocol and Custom TaqMan Gene Expression Assay protocol.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
After shipment, the assay mixes should be stored at -15 C to -20 C to maximize stability. Freeze/thaws should be kept to a minimum (a maximum of 10 freeze-thaw cycles is recommended). Keep all TaqMan and Custom TaqMan Gene Expression and SNP Genotyping Assays protected from direct exposure to light. Excessive exposure to light may affect the fluorescent probes. If desired, the Assay Mix may be diluted with TE buffer or DNase-free Water. It is recommended to make working stocks from the stock solutions provided in these kits.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
No, SYBR Green dye I is not compatible with the TaqMan Gene Expression Assays or Custom TaqMan Gene Expression Assays. TaqMan Gene Expression Assays or Custom TaqMan Gene Expression Assays contain TaqMan MGB probes combined with primers at non-limiting concentrations. The probes already have detection dyes conjugated to them, and SYBR Green is not needed. For more information, please consult the protocol of TaqMan Gene Expression Assays and Custom TaqMan Gene Expression Assays.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
The details for custom TaqMan assays can be downloaded at: https://www.thermofisher.com/order/taqman-files. Select the option "TaqMan Assays" and, in the screen that follows, indicate the sales order number and the Rack ID of your assay.
Both these numbers can be found on the assay package, next to the barcode.
TaqMan Gene Expression Assays are pre-designed, quantitative, human gene expression assays based on the TaqMan probe-based chemistry. The assays are formulated into a 20X mix.
Custom TaqMan Gene Expression Assays or Custom Plus TaqMan RNA Assays are designed based on the target sequences submitted by the customer, then synthesized, formulated into 20X concentration, and delivered. The target sequence information can be submitted by the customer through the online Custom Assay Design Tool. The integrity of the sequence information must be verified before the design process. When you submit a sequence, you have an option to let us run the bioinformatic evaluation of the target sequence to verify the integrity (Custom Plus) or you can run the bioinformatic evaluation yourself. This integrity verification can be accomplished through BLAST analysis or other methods of verifying sequence integrity. See the Custom TaqMan Assays Design and Ordering Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_042307.pdf) for more information.
Yes. For duplexing, we have VIC-labeled probes with primer limited concentrations (VIC PL) available from our pre-designed assay selection. The primer limited assay is used to detect the most abundant gene. For guidance on multiplexing, see the TaqMan Multiplex PCR Optimization User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/taqman_optimization_man.pdf).
Each TaqMan Gene Expression Assay consists of two unlabeled PCR primers and a FAM or VIC dye-labeled TaqMan MGB probe (with non-fluorescent quencher). All the components are combined into a 20X formulation. Purchase of a TaqMan Gene Expression Assay does not provide TaqMan PCR Master Mix.
Please review the following possible causes and solutions:
-The sample evaporated. Check the seal of the adhesive film for leaks.
- The well is empty because of inaccurate pipetting. Check the calibration of the pipettes, and pipette at least 5 µL of sample.
- The well is assigned a sample or target in the plate document or experiment, but the well is empty. Ensure that the plate document or experiment is set up correctly. Then try excluding the well and reanalyzing the data.
A small ΔRn can mean that the PCR efficiency was poor. Ensure that the reagents were used at the correct concentration. Alternatively, the quantity of the cDNA may be low (a low copy number of the target). In this case, we recommend that you increase the quantity of the cDNA in the reaction.
Most likely fluorescence did not stabilize to the buffer conditions of the reaction mix.
Note: This condition does not affect PCR or the final results.
We recommend that you try the following:
- Reset the lower value of the baseline range.
- Use an automatic baseline.
- Use the relative threshold algorithm (Crt). See Introduction to Gene Expression Getting Started Guide (Pub. No. 4454239).
Please review the following possible causes and solutions:
- The reagents were not mixed properly. We suggest increasing the length of time that you mix the reagents and confirming your mixing process by running a replicate assay.
- Pipetting was inaccurate. We recommend that you check the pipette calibration, and pipette at least 5 µL of sample to prepare the reaction mix.
- The threshold was not set correctly. Set the threshold above the noise level and where the replicates are tightest. Please see your real‑time PCR system user documentation for more information about setting the threshold.
- There was a low concentration of the target of interest. Rerun the assay with more cDNA template.
Please review the following possible causes and solutions:
- The endogenous control is not consistently expressed across the samples. Please ensure that the endogenous control is consistently expressed in your sample type.
- The sample concentrations vary. We recommend that you quantify and normalize the PCR samples before running the assay.
- Pipetting was inaccurate. We recommend that you check the pipette calibration, and pipet at least 5 µL of sample to prepare the reaction mix.
Most likely the reagents are contaminated with gDNA, amplicon or plasmid clones. We recommend that you clean your workspace and equipment, then rerun the assay using new reagents. We also recommend that you run no-RT controls to rule out genomic DNA contamination, and treat the sample with DNase.
Most likely the ROX dye was not set as the passive reference. Set ROX dye as the passive reference, then reanalyze the data.
Most likely incorrect dyes were selected for each target. We suggest checking the dyes selected for each target, then reanalyzing the data.
The sample evaporated. We recommend that you check the seal of the adhesive film for leaks before running the plate on the real-time PCR instrument.
It is possible that there was precipitation in the buffers. Before preapring reactions, we recommend that you mix the Master Mix thoroughly to produce a homogenous solution.
It as also possible that the reagents have degraded. Ensure that the kits and reagents have been stored according to the instructions on the packaging and that they have not expired.
Please review the following possible causes and solutions:
- The gene is not expressed in the sample. First, confirm that the gene is expressed in the sample type or tissue type at ncbi.nlm.nih.gov/unigene. If the gene is expressed, confirm the results by rerunning the sample using the same assay and/or rerunning the experiment using more of the sample. Avoid preparing PCR reaction mixes with more than 20% reverse transcription reaction. You can also run the experiment using an alternative assay, if available, that detects a different transcript or more than one transcript from the same gene.
Note: If the recommended actions do not resolve the problem, the result may be correct.
- The sample does not have enough copies of the target RNA. To confirm the results, we suggest rerunning the sample using the same assay and/or rerunning the assay using more of the sample. Avoid PCR reaction mix with more than 20% from the reverse transcription reaction.
Note: If the recommended actions do not resolve the problem, the result may be correct.
- One or more of the reaction components was not added. Please check your pipetting equipment and/or technique.
- Incorrect dyes were selected for each target. We recommend checking the dyes selected for each target, then reanalyze the data.
Please review the following possible causes and solutions:
- The baseline was set improperly. See your real‑time PCR system user guide for procedures on setting the baseline. We recommend switching from an automatic baseline to a manual baseline (or vice versa) and/or increasing the upper or lower value of the baseline range.
- The sample quality was poor. We recommend performing a quality check on the sample, then re-extracting the sample if needed.
- There were different concentrations caused my imprecise pipetting. Please follow accurate pipetting practices.
- The reagents or equipment are contaminated. Please ensure that your workspace and equipment are cleaned properly.
Most likely little or no MasterMix is present in the reaction due to inaccurate pipetting. Please follow accurate pipetting practices when setting up reactions.
Please review the following possible causes and solutions:
- The baseline was set too high and some samples have Ct values lower than the baseline stop value. We recommend that you switch from manual to automatic baselining, or move the baseline stop value to a lower Ct. The baseline stop value should be set to a Ct 2 cycles before the amplification curve crosses the threshold. Please see your real‑time PCR system user guide for procedures on setting the baseline.
-No baseline can be set because the amplification signal is detected too early in the PCR cycles. Diluting the sample can increase the Ct value.
Please review the following possible causes and solutions:
-Genomic DNA (gDNA) contamination occured. We recommend that you improve sample extraction methods and use DNase to ensure minimal gDNA contamination of the RNA.
For custom assays, we recommend that you design an assay that spans an exon-exon boundary. Please see the Custom TaqMan Assays Design and Ordering Guide (Pub. No. 4367671).
- The cDNA template or amplicon is contaminated. In this case, please follow established PCR good laboratory practices.
Please review the following possible causes and solutions:
- Contamination occurred. We recommend that you run a no-RT control to confirm that there was genomic DNA (gDNA) contamination. We also recommend the use DNase to ensure minimal gDNA contamination of the RNA.
For custom assays, we recommend that you design an assay that spans an exon-exon boundary. Please see the Custom TaqMan Assays Design and Ordering Guide (Pub. No. 4367671).
- Too much cDNA template was added to the reaction. We recommend that you quantitate the RNA before the reverse transcription (RT) reaction. After the RT reaction, adjust the concentration of cDNA before adding it to the reaction.
- The cDNA template or the amplicon is contaminated. Follow established PCR good laboratory practices.
Please review the following possible causes and solutions:
- One or more of the reaction components was not added. Ensure that the cDNA, the assay and the Master Mix were added to the reaction plate. The passive reference fails if the Master Mix is missing.
- Incorrect dyes were selected for each target. Check the dyes selected for each target, then reanalyze the data.
- The annealing temperature was too high for the primers and/or probe. Ensure that the correct annealing and extension temperatures are set, and that the real-time PCR instrument is calibrated and maintained regularly.
- Inappropriate reaction conditions were used. Ensure that the properties and the thermal protocol are correct, then troubleshoot the real-time PCR optimization.
- The template is degraded. Determine the quality of the template, then rerun the assay with fresh template if needed. We recommend that you use use RNase-free reagents and an RNase inhibitor.
- Inhibitors are present in the reaction. To check for inhibitors, we recommend that you run a serial dilution of your sample with an expressing assay (for example, an endogenous control). If an inhibitor is present, the high concentration samples yield higher-than-expected Ct values because the samples are not diluted.
- The baseline and/or threshold was improperly set. See your real-time PCR system user guide for procedures on setting the baseline and threshold. Some possible solutions to this issue are switching from an automatic baseline to a manual baseline (or vice versa), and/or lowering the threshold value to fall within the appropriate range.
- The reverse transcription failed. We recommend checking the RNA integrity and concentration, checking for RNase activity, following the recommended thermal profile, and/or repeating the reverse transcription using new reagents.
- (Custom TaqManGene Expression Assays only) The design or synthesis of the custom assay failed. Ensure that the sequence you submitted is correct. We also recomend that you check for an alternative trascript or splice variant.
- (Custom TaqManGene Expression Assays only) The assay is designed in a variable regions of the gene transccript. Ensure that the location that is targeted by the assay is not within the 5' untranslated region (UTR), which can be highly variable
between transcripts. If the assay is designed within the 5' UTR, we recommend that you select a different assay that is within the coding region of the transcript. Otherwise, select an assay for an alternative transcriptor splice variant.
The sequence provided for the assay design may contain mismatches with sample sequences. We recommend that you perform bioinformatics analysis for assay design.
For more information, see the Custom TaqMan Assays Design and Ordering Guide (Pub.No. 4367671).
There may be interaction between the primer and probe. We recommend that you adjust the threshold manually, or select another assay from the same gene, if available.
For orders placed after March 2016, assay information files can be accessed from this page: www.thermofisher.com/taqmanfiles
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
The number of reactions will depend on your final reaction volume. The Custom and Custom Plus TaqMan RNA assays are supplied with the following volumes:
Small: 360 µL (20X)
Medium: 750 µL (20X)
Large: 976 µL (60X)
Assuming a 20 µL final reaction volume, the small size would provide 360 reactions, the medium size would provide 750 reactions, and the large size would provide 2,900 reactions.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
The Custom and Custom Plus TaqMan RNA assays are supplied with the following volumes:
Small: 360 µL
Medium: 750 µL
Large: 976 µL
Please note that the large scale is supplied in a 60X format, while the small and medium scales are supplied in 20X format.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
Custom and Custom Plus TaqMan RNA assays can be ordered online with either FAM, VIC, or VIC primer limited.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
Custom and Custom Plus TaqMan RNA assays can be ordered online with either FAM or VIC dye, with a MGB-NFQ quencher.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.
Yes, we offer two options for obtaining endogenous control assays for gene expression studies:
The first option is our specially designed TaqMan Endogenous Controls products, which are a collection of probe and primer sets that are optimized, pre-formulated, and ready-to-use specifically as control assays to save on time. Endogenous Control formulations are available as primer limited and contain probes labeled with FAM or VIC reporter dyes. This allows multiplexing of TaqMan Endogenous Controls with TaqMan target gene primer and probe sets, provided that the control gene is more abundantly expressed than the target gene.
Another option is to simply order a general TaqMan Gene Expression Assay for genes that are commonly used as endogenous controls, such as 18S rRNA, GAPDH, beta-actin, etc. The TaqMan Gene Expression Assays are biologically informative, pre-formulated assays for rapid, reliable detection and quantification of human mRNA transcripts. They come in a wide variety of reaction sizes and formats, and may be ordered with either a FAM or VIC reporter dye label with MGB quenchers. You can also specify that a VIC-labeled probe should be Primer Limited (PL) for multiplexing.