Real-Time PCR Troubleshooting

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en-us LUSOBDHYP Troubleshooting Real-Time PCR Troubleshooting /webroot/web/html/lsr/solutions/technologies/qpcr_real-time_pcr <script type="text/javascript"><!-- $('head').append('<meta name="DCSext.at_banner" content="Real-Time PCR Troubleshooting" /><meta name="DCSext.at_banner_e" content="v" />'); $(document).ready(function () { $("#rightcol").append('<div id="relatedProducts">&nbsp;</div>'); $('#relatedProducts').load('/webroot/web/html/lsr/solutions/technologies/pcr/related-products-a-t-rtpcr-troubleshooting.html'); }); // --></script> <p>Are you having trouble with your real-time PCR experiment? Do you need help diagnosing your experiment? The <strong>Real-Time PCR Doctor</strong> is here to help. Unexpected fluorescence data are symptomatic of problems with your real-time PCR reaction components or amplification protocol. Click one of the symptoms below to learn about possible causes and treatments.</p> <p><strong>Related Topics:</strong> <a href="/evportal/destination/solutions?catID=LUSO4W8UU#1">What Is Real-Time PCR?</a>, <a href="/evportal/destination/solutions?catID=LUSO4W8UU#2">How Real-Time PCR Works</a>, and <a href="/evportal/destination/solutions?catID=LUSO7RIVK">qPCR Assay Design and Optimization</a>. <br /></p> <p>&nbsp;</p> <p>&nbsp;</p> <p>&nbsp;</p> <p>&nbsp;</p> <p><br /></p> <div id="didYouknow"> <script type="text/javascript"><!-- $('#didYouknow').load('/webroot/web/html/lsr/products/amplification_pcr/did-you-know-primepcr.html'); // --></script> </div> <div class="contentroundwrap"> <div class="contentroundtop"> <h3>Gene Expression/Quantification Experiments:</h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><strong>My experiment displays:</strong></p> <table class="pd_table pd_centeredcell" border="0"> <tbody> <tr> <td width="25%"><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel1"> <img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img1.jpg" border="0" alt="Poor Signal or No Signal" width="110" height="110" /></a><br /> <a href="/evportal/destination/solutions?catID=LUSOCICZF#gel1">Poor signal or no signal</a><br />&nbsp;</td> <td width="25%"><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel2"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img2.jpg" border="0" alt="Signal in Negative Control" width="110" height="110" /></a><br /> <a href="/evportal/destination/solutions?catID=LUSOCICZF#gel2">Signal in negative control</a><br />&nbsp;</td> <td width="25%"><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel3"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img3.jpg" border="0" alt="CT Values Differing Across Replicate Samples" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel3">Poor reproducibility across replicate samples</a></td> <td width="25%"><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel4"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img4.jpg" border="0" alt="Low or High Reaction Efficiency" width="110" height="110" /></a><br /> <a href="/evportal/destination/solutions?catID=LUSOCICZF#gel4">Low or high reaction efficiency</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"> <div class="contentroundtop"> <h3>Allelic Discrimination Experiments</h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><strong>My experiment displays:</strong></p> <table class="pd_table pd_centeredcell" border="0"> <tbody> <tr> <td width="50%"><a href="/evportal/destination/solutions?catID=LUSOFGDN#gel1"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img5.jpg" border="0" alt="More than 3 clusters" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOFGDN#gel1">More than 3 clusters</a></td> <td width="50%"><a href="/evportal/destination/solutions?catID=LUSOFGDN#gel2"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img6.jpg" border="0" alt="Homozygotes as heterozygotes" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOFGDN#gel2">Homozygotes as heterozygotes</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"> <div class="contentroundtop"> <h3><strong>High Resolution Melt (HRM) Experiments</strong>:</h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><strong>My experiment displays:</strong></p> <table class="pd_table pd_centeredcell" border="0"> <tbody> <tr> <td width="32%"><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel1"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img7.jpg" border="0" alt="Missed call or clustering" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel1">Missed call or clustering</a></td> <td width="32%"><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel2"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img8.jpg" border="0" alt="Amplification did not reach plateau" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel2">Amplification did not reach plateau</a></td> <td width="32%"><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel3"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img9.jpg" border="0" alt="Poor amplification or failed reaction" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel3">Poor amplification or failed reaction</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <p style="margin-top: 10px;">If you don't see your symptom listed above, please contact technical support at 1-800-424-6723, option 2.</p> <br /> <a name="further_reading"></a> <h3>Further Reading</h3> <img style="height: 1px; margin: 5px 0px 15px; width: 620px;" src="http://www.bio-rad.com/webroot/web/images/1x1grey.gif" alt="" /> <p>Hu CJ et al. (2006). Differential regulation of the transcriptional activities of hypoxia-inducible factor 1 alpha (HIF-1alpha) and HIF-2alpha in stem cells. Mol Cell Biol 26, 3514&ndash;3526. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16611993" target="_blank">16611993</a></p> <p>Ivanova N et al. (2006). Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533&ndash;538. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16767105" target="_blank">16767105</a></p> <p>Li Y et al. (2006). DAPK1 variants are associated with Alzheimer's disease and allele-specific expression. Hum Mol Genet 15, 2560&ndash;2568. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16847012" target="_blank">16847012</a></p> <p>Morozumi M et al. (2006). Simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time PCR with pathogen-specific molecular beacon probes. J Clin Microbiol. 44, 1440&ndash;1446. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16597874" target="_blank">16597874</a></p> <p>Ridd K et al. (2006). Association of gene expression with sequential proliferation, differentiation, and tumor formation in murine skin. Carcinogenesis 27, 1556&ndash;1566. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16537558" target="_blank">16537558</a></p> <div class="top"><a href="#helptop">Back to Top</a></div> <p> <script src="http://admin.brightcove.com/js/BrightcoveExperiences.js" type="text/javascript"></script> </p> <div class="videowrap"> <div class="videoImg"><a title="PCR Fundamentals" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_pcr_fundamentals.html' );" href="javascript:void(0);"><img style="border:none" src="/webroot/web/images/lsr/support/tutorials/global/ov_pcr_fundamentals.jpg" alt="PCR Fundamentals" /></a></div> <div class="videoDesc"><br /><a title="PCR Fundamentals" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_pcr_fundamentals.html' );" href="javascript:void(0);">PCR Fundamentals</a><br /> &nbsp;<br />&nbsp;</div> <div class="clear">&nbsp;</div> </div> <p> <script type="text/javascript"><!-- brightcove.createExperiences(); // --></script> </p> <div class="videowrap"> <div class="videoImg"><a title="Performing Fast PCR" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_fast_pcr.html' );" href="javascript:void(0);"><img style="border:none" src="/webroot/web/images/lsr/support/tutorials/global/ov_fast_pcr.jpg" alt="Performing Fast PCR" /></a></div> <div class="videoDesc"><br /><a title="Performing Fast PCR" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_fast_pcr.html' );" href="javascript:void(0);">Performing Fast PCR</a><br /> &nbsp;<br />&nbsp;</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap"> <div class="videoImg"><a title="Quantitative PCR (qPCR): Validating a SYBR Green I Assay" onclick="window.open('/webroot/web/html/lsr/support/qPCR_Assay_Validation.htm', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);"><img style="border:none;" src="/webroot/web/images/lsr/support/tutorials/global/qpcr_validating_qybr_green.png" alt="" /></a></div> <div class="videoDesc"><a title="Quantitative PCR (qPCR): Validating a SYBR Green I Assay" onclick="window.open('/webroot/web/html/lsr/support/qPCR_Assay_Validation.htm', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);">Quantitative PCR (qPCR): Validating a SYBR<sup>&reg;</sup> Green I Assay</a><br /> This tutorial describes the characteristics of an optimized SYBR Green I quantitative PCR (qPCR) assay. qPCR assays must be optimized to ensure results that are biologically and statistically significant. Topics include a brief review of qPCR chemistry, with an emphasis on SYBR Green I reactions, and definitions of the four main characteristics, or hallmarks, of an optimized qPCR assay.</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap"> <div class="videoImg"><a title="Quantitative PCR (qPCR): Hallmarks of an Optimized SYBR Green I Assay" onclick="window.open('/webroot/web/movies/lsr/support/qPCR_Hallmarks.htm', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);"><img style="border:none;" src="/webroot/web/images/lsr/support/tutorials/global/qpcr_hallmark.png" alt="" /></a></div> <div class="videoDesc"><a title="Quantitative PCR (qPCR): Hallmarks of an Optimized SYBR Green I Assay" onclick="window.open('/webroot/web/movies/lsr/support/qPCR_Hallmarks.htm', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);">Quantitative PCR (qPCR): Hallmarks of an Optimized SYBR Green I Assay</a><br /> This tutorial describes the characteristics of an optimized SYBR<sup>&reg;</sup> Green I quantitative PCR (qPCR) assay. qPCR assays must be optimized to ensure results that are biologically and statistically significant. Topics include a brief review of qPCR chemistry, with an emphasis on SYBR Green I reactions, and definitions of the four main characteristics, or hallmarks, of an optimized qPCR assay.</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap vwrap_last"> <div class="videoImg"><a title="Data Analysis: Real-Time Quantitative PCR" onclick="window.open('http://bio-rad.centra.com/bio-radroot/learning%20objects/data_analysis/data_analysis.html', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);"><img style="border:none;" src="http://www.biorad-ads.com/solutions/webroot/web/images/lsr/support/tutorials/global/ov_data_analysis_qpcr.gif" alt="" /></a></div> <div class="videoDesc"><a title="Data Analysis: Real-Time Quantitative PCR" onclick="window.open('http://bio-rad.centra.com/bio-radroot/learning%20objects/data_analysis/data_analysis.html', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);">Data Analysis: Real-Time Quantitative PCR</a><br />This tutorial pertains to the analysis of real-time quantitative PCR (qPCR) data.</div> <div class="clear">&nbsp;</div> </div> 5692 Real-Time qPCR as a Tool for Evaluating RNAi-Mediated Gene Silencing, Rev A 5692 /webroot/web/pdf/lsr/literature/Bulletin_5692.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Real-Time qPCR as a Tool for Evaluating RNAi-Mediated Gene Silencing, Rev A (tech note) 5692 sirna, polymerase chain reaction, interference, knockdown, mycn, rna, knock down, neuroblastoma, pcr, rt-qpcr, cancer, rt qpcr, rtqpcr, genes, LIT5692, real time, si rna, tp53, neuro blastoma, quantitative, realtime 6004 A Practical Guide to High Resolution Melt Analysis Genotyping, Rev A 6004 /webroot/web/pdf/lsr/literature/Bulletin_6004.pdf Literature PDF Manuals_and_Quick_Guides /webroot/web/images/general/icons/icon_pdf.gif No A Practical Guide to High Resolution Melt Analysis Genotyping, Rev A 6004 6004, amplicon, CFX96 real-time PCR detection system, CFX384, DNA melt curve profiles, genetic variation, genome sequencing, high-throughput, high throughput, HRM, polymerase chain reaction, Precision Melt Analysis software, primer design, quantitative PCR, qPCR, SNP profiling, 185-5096, 1855096, 185-5384, 1855384, 184-5025, 1845025 6009 High Resolution Melt Parameter Considerations for Optimal Data Resolution, Rev A 6009 /webroot/web/pdf/lsr/literature/Bulletin_6009.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No High Resolution Melt Parameter Considerations for Optimal Data Resolution, Rev A 6009 6009, amplicon, CFX96 real-time PCR detection system, CFX384, DNA methylation analysis, genetic variation, genome sequencing, HRM, HRMA, mutation scanning, Precision Melt Analysis software, reaction optimization, SNP profiling, species identification, 184-5025, 1845025, 185-5096, 1855096, 185-5384, 1855384 6047 Optical Design of CFX96&trade; Real-Time PCR Detection System Eliminates the Requirement of a Passive Reference Dye, Rev A 6047 /webroot/web/pdf/lsr/literature/Bulletin_6047A.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Optical Design of CFX96&trade; Real-Time PCR Detection System Eliminates the Requirement of a Passive Reference Dye, Rev A 6047 6047, ABI 7500, Applied Biosystems, CFX, CFX384, Cq, fluorescence measurements, iQ supermix, iScript cDNA synthesis kit, polymerase chain reaction, quantification cycle, quantitative PCR, qPCR, realtime, real time, ROX normalization, 185-5096, 1855096, 185-5384, 1855384 5962 Fast Optimization of a Multiplex Influenza Identification Panel Using a Thermal Gradient, Rev A 5962 /webroot/web/pdf/lsr/literature/Bulletin_5962A.pdf Literature PDF Manuals_and_Quick_Guides /webroot/web/images/icon_pdf.gif No Fast Optimization of a Multiplex Influenza Identification Panel Using a Thermal Gradient, Rev A 5962 5’ nuclease probe-based assay, CFX96 real-time PCR detection system, epidemiology, flu A, B, H1N1, H3N2, iScript one-step RT-PCR kit, optimize, pandemic, qPCR, reverse transcription, RT-qPCR, sensitivity, swine, TaqMan, 170-8894, 1708894, 170-8895, 1708895, 184-5096, 1845096, 185-5096, 1855096 Related Websites <ul class="rightarrow"> <li><a class="subaccord" href="http://evolution.gene-quantification.info/" target="_blank">Real-Time PCR</a></li> </ul> Life Science Research/Products/Nucleic Acid Sample Preparation and Purification/RNA Isolation/Aurum Total RNA 96 Kit ->MT::49da5896-dfde-4292-8021-9df1d8b77fec##Life Science Research/Products/Nucleic Acid Sample Preparation and Purification/DNA Isolation/Quantum Prep Plasmid Kits ->MT::7167c5db-d618-47a5-9185-9c612fe4fbe0##Life Science Research/Products/Amplification - PCR/PCR Reagents/Reverse Transcription Reagents/iScript Advanced cDNA Synthesis Kit ->MTS::LIXR5J15##Life Science Research/Products/Amplification - PCR/PCR Reagents/Supermixes for PCR and Real-Time PCR/SsoAdvanced SYBR Green Supermix ->MTS::LML5JY15##Life Science Research/Products/Amplification - PCR/PCR Reagents/Supermixes for PCR and Real-Time PCR/Precision Melt Supermix ->MTS::LNIQ3X15## Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/Assay Design and Optimization ->MTS::LUSO7RIVK##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/Reagents ->MTS::LUSO6XKSY##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/Data analysis ->MTS::LUSOA9LPT##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/High Resolution Melting -HRM- ->MTS::LUSOIH97Q##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/MIQE and RDML ->MTS::LUSO8N4EH## Eddie C Real-Time PCR Troubleshooting Having trouble with your real-time PCR experiments? The Real-Time PCR Doctor is here to help! pcr, pcr analysis, pcr troubleshooting, pcr doctor, pcr protocol, pcr reagents, pcr problems, qpcr, qpcr analysis, qpcr troubleshooting, qpcr protocol, qpcr reagents, qpcr problems, real-time pcr, real-time pcr analysis, real-time pcr troubleshooting, real-time pcr protocol, real-time pcr reagents, real-time pcr problems 01/06/12 10:17 AM 01/06/21 10:19 AM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Technologies/qPCR_Real_Time_PCR N 0 Real-Time PCR Doctor | Gene Expression/Quantification Experiments /en-us/applications-technologies/applications-technologies/real-time-pcr-doctor-gene-expression-quantification-experiments?ID=LUSOCICZF Real-Time PCR Doctor | Allelic Discrimination Experiments /en-us/applications-technologies/applications-technologies/real-time-pcr-doctor-allelic-discrimination-experiments?ID=LUSOFGDN Real-Time PCR Doctor | High Resolution Melt (HRM) Analysis Experiments /en-us/applications-technologies/applications-technologies/real-time-pcr-doctor-high-resolution-melt-hrm-analysis-experiments?ID=LUSOGO2B7 What is Real-Time PCR (qPCR)? /en-us/applications-technologies/applications-technologies/real-time-pcr-troubleshooting?ID=LUSO4W8UU

Are you having trouble with your real-time PCR experiment? Do you need help diagnosing your experiment? The Real-Time PCR Doctor is here to help. Unexpected fluorescence data are symptomatic of problems with your real-time PCR reaction components or amplification protocol. Click one of the symptoms below to learn about possible causes and treatments.

Related Topics: What Is Real-Time PCR?, How Real-Time PCR Works, and qPCR Assay Design and Optimization.

 

 

 

 


Allelic Discrimination Experiments

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High Resolution Melt (HRM) Experiments:

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If you don't see your symptom listed above, please contact technical support at 1-800-424-6723, option 2.


Further Reading

Hu CJ et al. (2006). Differential regulation of the transcriptional activities of hypoxia-inducible factor 1 alpha (HIF-1alpha) and HIF-2alpha in stem cells. Mol Cell Biol 26, 3514–3526. PMID: 16611993

Ivanova N et al. (2006). Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533–538. PMID: 16767105

Li Y et al. (2006). DAPK1 variants are associated with Alzheimer's disease and allele-specific expression. Hum Mol Genet 15, 2560–2568. PMID: 16847012

Morozumi M et al. (2006). Simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time PCR with pathogen-specific molecular beacon probes. J Clin Microbiol. 44, 1440–1446. PMID: 16597874

Ridd K et al. (2006). Association of gene expression with sequential proliferation, differentiation, and tumor formation in murine skin. Carcinogenesis 27, 1556–1566. PMID: 16537558

 
 
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    This tutorial describes the characteristics of an optimized SYBR Green I quantitative PCR (qPCR) assay. qPCR assays must be optimized to ensure results that are biologically and statistically significant. Topics include a brief review of qPCR chemistry, with an emphasis on SYBR Green I reactions, and definitions of the four main characteristics, or hallmarks, of an optimized qPCR assay.
     
    Quantitative PCR (qPCR): Hallmarks of an Optimized SYBR Green I Assay
    This tutorial describes the characteristics of an optimized SYBR® Green I quantitative PCR (qPCR) assay. qPCR assays must be optimized to ensure results that are biologically and statistically significant. Topics include a brief review of qPCR chemistry, with an emphasis on SYBR Green I reactions, and definitions of the four main characteristics, or hallmarks, of an optimized qPCR assay.
     
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    Literature
    Number Description Download
    5692 Real-Time qPCR as a Tool for Evaluating RNAi-Mediated Gene Silencing, Rev A Click to download
    6004 A Practical Guide to High Resolution Melt Analysis Genotyping, Rev A Click to download
    6009 High Resolution Melt Parameter Considerations for Optimal Data Resolution, Rev A Click to download
    6047 Optical Design of CFX96&trade; Real-Time PCR Detection System Eliminates the Requirement of a Passive Reference Dye, Rev A Click to download
    5962 Fast Optimization of a Multiplex Influenza Identification Panel Using a Thermal Gradient, Rev A Click to download
     
     
    LUSOBDHYP [referer] = [http://www.bio-rad.com/en-us/applications-technologies/real-time-pcr-troubleshooting?ID=LUSOBDHYP] [x-forwarded-proto] = [http] [accept-language] = [en-ca] [x-forwarded-port] = [80] [x-forwarded-for] = [131.215.220.161, 10.232.18.193] [dnt] = [1] [accept] = [text/html,application/xhtml+xml,application/xml;q=0.9,*/*;q=0.8] [seourl] = [/en-us/applications-technologies/real-time-pcr-troubleshooting] [x-amzn-trace-id] = [Root=1-5b78f124-016e54d6ba979eecb063eaef] [x-forwarded-server] = [lsds-prod-s.br.aws-livesite.io] [x-forwarded-host] = [www.bio-rad.com] [x-query-string] = [ID=LUSOBDHYP] [host] = [10.232.16.28:1776] [upgrade-insecure-requests] = [1] [x-request-uri] = [/en-us/applications-technologies/real-time-pcr-troubleshooting] [connection] = [Keep-Alive] [accept-encoding] = [gzip, deflate] [user-agent] = [Mozilla/5.0 (iPhone; CPU iPhone OS 11_4_1 like Mac OS X) AppleWebKit/605.1.15 (KHTML, like Gecko) Version/11.0 Mobile/15E148 Safari/604.1] AppTech/AppTechDetails pageStyleKey internet/solutions_sub applications-technologies/real-time-pcr-troubleshooting LSR LUSOBDHYP Troubleshooting Real-Time PCR Troubleshooting /webroot/web/html/lsr/solutions/technologies/qpcr_real-time_pcr <script type="text/javascript"><!-- $('head').append('<meta name="DCSext.at_banner" content="Real-Time PCR Troubleshooting" /><meta name="DCSext.at_banner_e" content="v" />'); $(document).ready(function () { $("#rightcol").append('<div id="relatedProducts">&nbsp;</div>'); $('#relatedProducts').load('/webroot/web/html/lsr/solutions/technologies/pcr/related-products-a-t-rtpcr-troubleshooting.html'); }); // --></script> <p>Are you having trouble with your real-time PCR experiment? Do you need help diagnosing your experiment? The <strong>Real-Time PCR Doctor</strong> is here to help. Unexpected fluorescence data are symptomatic of problems with your real-time PCR reaction components or amplification protocol. Click one of the symptoms below to learn about possible causes and treatments.</p> <p><strong>Related Topics:</strong> <a href="/evportal/destination/solutions?catID=LUSO4W8UU#1">What Is Real-Time PCR?</a>, <a href="/evportal/destination/solutions?catID=LUSO4W8UU#2">How Real-Time PCR Works</a>, and <a href="/evportal/destination/solutions?catID=LUSO7RIVK">qPCR Assay Design and Optimization</a>. <br /></p> <p>&nbsp;</p> <p>&nbsp;</p> <p>&nbsp;</p> <p>&nbsp;</p> <p><br /></p> <div id="didYouknow"> <script type="text/javascript"><!-- $('#didYouknow').load('/webroot/web/html/lsr/products/amplification_pcr/did-you-know-primepcr.html'); // --></script> </div> <div class="contentroundwrap"> <div class="contentroundtop"> <h3>Gene Expression/Quantification Experiments:</h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><strong>My experiment displays:</strong></p> <table class="pd_table pd_centeredcell" border="0"> <tbody> <tr> <td width="25%"><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel1"> <img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img1.jpg" border="0" alt="Poor Signal or No Signal" width="110" height="110" /></a><br /> <a href="/evportal/destination/solutions?catID=LUSOCICZF#gel1">Poor signal or no signal</a><br />&nbsp;</td> <td width="25%"><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel2"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img2.jpg" border="0" alt="Signal in Negative Control" width="110" height="110" /></a><br /> <a href="/evportal/destination/solutions?catID=LUSOCICZF#gel2">Signal in negative control</a><br />&nbsp;</td> <td width="25%"><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel3"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img3.jpg" border="0" alt="CT Values Differing Across Replicate Samples" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel3">Poor reproducibility across replicate samples</a></td> <td width="25%"><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel4"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img4.jpg" border="0" alt="Low or High Reaction Efficiency" width="110" height="110" /></a><br /> <a href="/evportal/destination/solutions?catID=LUSOCICZF#gel4">Low or high reaction efficiency</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"> <div class="contentroundtop"> <h3>Allelic Discrimination Experiments</h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><strong>My experiment displays:</strong></p> <table class="pd_table pd_centeredcell" border="0"> <tbody> <tr> <td width="50%"><a href="/evportal/destination/solutions?catID=LUSOFGDN#gel1"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img5.jpg" border="0" alt="More than 3 clusters" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOFGDN#gel1">More than 3 clusters</a></td> <td width="50%"><a href="/evportal/destination/solutions?catID=LUSOFGDN#gel2"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img6.jpg" border="0" alt="Homozygotes as heterozygotes" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOFGDN#gel2">Homozygotes as heterozygotes</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"> <div class="contentroundtop"> <h3><strong>High Resolution Melt (HRM) Experiments</strong>:</h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><strong>My experiment displays:</strong></p> <table class="pd_table pd_centeredcell" border="0"> <tbody> <tr> <td width="32%"><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel1"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img7.jpg" border="0" alt="Missed call or clustering" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel1">Missed call or clustering</a></td> <td width="32%"><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel2"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img8.jpg" border="0" alt="Amplification did not reach plateau" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel2">Amplification did not reach plateau</a></td> <td width="32%"><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel3"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img9.jpg" border="0" alt="Poor amplification or failed reaction" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel3">Poor amplification or failed reaction</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <p style="margin-top: 10px;">If you don't see your symptom listed above, please contact technical support at 1-800-424-6723, option 2.</p> <br /> <a name="further_reading"></a> <h3>Further Reading</h3> <img style="height: 1px; margin: 5px 0px 15px; width: 620px;" src="http://www.bio-rad.com/webroot/web/images/1x1grey.gif" alt="" /> <p>Hu CJ et al. (2006). Differential regulation of the transcriptional activities of hypoxia-inducible factor 1 alpha (HIF-1alpha) and HIF-2alpha in stem cells. Mol Cell Biol 26, 3514&ndash;3526. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16611993" target="_blank">16611993</a></p> <p>Ivanova N et al. (2006). Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533&ndash;538. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16767105" target="_blank">16767105</a></p> <p>Li Y et al. (2006). DAPK1 variants are associated with Alzheimer's disease and allele-specific expression. Hum Mol Genet 15, 2560&ndash;2568. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16847012" target="_blank">16847012</a></p> <p>Morozumi M et al. (2006). Simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time PCR with pathogen-specific molecular beacon probes. J Clin Microbiol. 44, 1440&ndash;1446. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16597874" target="_blank">16597874</a></p> <p>Ridd K et al. (2006). Association of gene expression with sequential proliferation, differentiation, and tumor formation in murine skin. Carcinogenesis 27, 1556&ndash;1566. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16537558" target="_blank">16537558</a></p> <div class="top"><a href="#helptop">Back to Top</a></div> <p> <script src="http://admin.brightcove.com/js/BrightcoveExperiences.js" type="text/javascript"></script> </p> <div class="videowrap"> <div class="videoImg"><a title="PCR Fundamentals" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_pcr_fundamentals.html' );" href="javascript:void(0);"><img style="border:none" src="/webroot/web/images/lsr/support/tutorials/global/ov_pcr_fundamentals.jpg" alt="PCR Fundamentals" /></a></div> <div class="videoDesc"><br /><a title="PCR Fundamentals" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_pcr_fundamentals.html' );" href="javascript:void(0);">PCR Fundamentals</a><br /> &nbsp;<br />&nbsp;</div> <div class="clear">&nbsp;</div> </div> <p> <script type="text/javascript"><!-- brightcove.createExperiences(); // --></script> </p> <div class="videowrap"> <div class="videoImg"><a title="Performing Fast PCR" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_fast_pcr.html' );" href="javascript:void(0);"><img style="border:none" src="/webroot/web/images/lsr/support/tutorials/global/ov_fast_pcr.jpg" alt="Performing Fast PCR" /></a></div> <div class="videoDesc"><br /><a title="Performing Fast PCR" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_fast_pcr.html' );" href="javascript:void(0);">Performing Fast PCR</a><br /> &nbsp;<br />&nbsp;</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap"> <div class="videoImg"><a title="Quantitative PCR (qPCR): Validating a SYBR Green I Assay" onclick="window.open('/webroot/web/html/lsr/support/qPCR_Assay_Validation.htm', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);"><img style="border:none;" src="/webroot/web/images/lsr/support/tutorials/global/qpcr_validating_qybr_green.png" alt="" /></a></div> <div class="videoDesc"><a title="Quantitative PCR (qPCR): Validating a SYBR Green I Assay" onclick="window.open('/webroot/web/html/lsr/support/qPCR_Assay_Validation.htm', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);">Quantitative PCR (qPCR): Validating a SYBR<sup>&reg;</sup> Green I Assay</a><br /> This tutorial describes the characteristics of an optimized SYBR Green I quantitative PCR (qPCR) assay. qPCR assays must be optimized to ensure results that are biologically and statistically significant. Topics include a brief review of qPCR chemistry, with an emphasis on SYBR Green I reactions, and definitions of the four main characteristics, or hallmarks, of an optimized qPCR assay.</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap"> <div class="videoImg"><a title="Quantitative PCR (qPCR): Hallmarks of an Optimized SYBR Green I Assay" onclick="window.open('/webroot/web/movies/lsr/support/qPCR_Hallmarks.htm', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);"><img style="border:none;" src="/webroot/web/images/lsr/support/tutorials/global/qpcr_hallmark.png" alt="" /></a></div> <div class="videoDesc"><a title="Quantitative PCR (qPCR): Hallmarks of an Optimized SYBR Green I Assay" onclick="window.open('/webroot/web/movies/lsr/support/qPCR_Hallmarks.htm', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);">Quantitative PCR (qPCR): Hallmarks of an Optimized SYBR Green I Assay</a><br /> This tutorial describes the characteristics of an optimized SYBR<sup>&reg;</sup> Green I quantitative PCR (qPCR) assay. qPCR assays must be optimized to ensure results that are biologically and statistically significant. Topics include a brief review of qPCR chemistry, with an emphasis on SYBR Green I reactions, and definitions of the four main characteristics, or hallmarks, of an optimized qPCR assay.</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap vwrap_last"> <div class="videoImg"><a title="Data Analysis: Real-Time Quantitative PCR" onclick="window.open('http://bio-rad.centra.com/bio-radroot/learning%20objects/data_analysis/data_analysis.html', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);"><img style="border:none;" src="http://www.biorad-ads.com/solutions/webroot/web/images/lsr/support/tutorials/global/ov_data_analysis_qpcr.gif" alt="" /></a></div> <div class="videoDesc"><a title="Data Analysis: Real-Time Quantitative PCR" onclick="window.open('http://bio-rad.centra.com/bio-radroot/learning%20objects/data_analysis/data_analysis.html', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);">Data Analysis: Real-Time Quantitative PCR</a><br />This tutorial pertains to the analysis of real-time quantitative PCR (qPCR) data.</div> <div class="clear">&nbsp;</div> </div> 5692 6004 6009 6047 5962 Related Websites <ul class="rightarrow"> <li><a class="subaccord" href="http://evolution.gene-quantification.info/" target="_blank">Real-Time PCR</a></li> </ul> Life Science Research/Products/Nucleic Acid Sample Preparation and Purification/RNA Isolation/Aurum Total RNA 96 Kit ->MT::49da5896-dfde-4292-8021-9df1d8b77fec##Life Science Research/Products/Nucleic Acid Sample Preparation and Purification/DNA Isolation/Quantum Prep Plasmid Kits ->MT::7167c5db-d618-47a5-9185-9c612fe4fbe0##Life Science Research/Products/Amplification - PCR/PCR Reagents/Reverse Transcription Reagents/iScript Advanced cDNA Synthesis Kit ->MTS::LIXR5J15##Life Science Research/Products/Amplification - PCR/PCR Reagents/Supermixes for PCR and Real-Time PCR/SsoAdvanced SYBR Green Supermix ->MTS::LML5JY15##Life Science Research/Products/Amplification - PCR/PCR Reagents/Supermixes for PCR and Real-Time PCR/Precision Melt Supermix ->MTS::LNIQ3X15## Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/Assay Design and Optimization ->MTS::LUSO7RIVK##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/Reagents ->MTS::LUSO6XKSY##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/Data analysis ->MTS::LUSOA9LPT##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/High Resolution Melting -HRM- ->MTS::LUSOIH97Q##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/MIQE and RDML ->MTS::LUSO8N4EH## Eddie C Real-Time PCR Troubleshooting Having trouble with your real-time PCR experiments? The Real-Time PCR Doctor is here to help! pcr, pcr analysis, pcr troubleshooting, pcr doctor, pcr protocol, pcr reagents, pcr problems, qpcr, qpcr analysis, qpcr troubleshooting, qpcr protocol, qpcr reagents, qpcr problems, real-time pcr, real-time pcr analysis, real-time pcr troubleshooting, real-time pcr protocol, real-time pcr reagents, real-time pcr problems 01/06/12 10:17 AM 01/06/21 10:19 AM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Technologies/qPCR_Real_Time_PCR N 0

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