Fast qPCR assayoptimization and validation    techniques for HTS        Francisco Bizouarn        International Field Appl...
Generating a good assay is easyAMPLIFICATION                                • Following a few simple steps:               ...
Assay designAMPLIFICATION           • Often oversimplified by the use of software or by             many companies that of...
CCL26 cDNA sequenceAMPLIFICATION                CTGGAATTGA   GGCTGAGCCA   AAGACCCCAG        GGCCGTCTCA   GTCTCATAAA       ...
Sequence Alignment (BLAST)AMPLIFICATION                                          •   Prior to designing primers, it’s     ...
CCL26 with homologous sequencesAMPLIFICATION                CTGGAATTGA   GGCTGAGCCA   AAGACCCCAG        GGCCGTCTCA   GTCTC...
CCL26 with homologous sequencesAMPLIFICATION                CTGGAATTGA   GGCTGAGCCA   AAGACCCCAG        GGCCGTCTCA   GTCTC...
2nd structure analysis of CCL26AMPLIFICATION                                          •   DNA is often seen as a linear   ...
CCL26 with 2nd structuresAMPLIFICATION                CTGGAATTGA   GGCTGAGCCA   AAGACCCCAG        GGCCGTCTCA   GTCTCATAAA ...
CCL26 with 2nd structuresAMPLIFICATION                CTGGAATTGA   GGCTGAGCCA   AAGACCCCAG        GGCCGTCTCA   GTCTCATAAA ...
Amplicon sizeAMPLIFICATION           • Classic qPCR rules dictate that amplification products be             between 75 an...
Design primersAMPLIFICATION                                          •    Some primer design packages will                ...
CCL26 primer designAMPLIFICATION                CTGGAATTGA   GGCTGAGCCA   AAGACCCCAG        GGCCGTCTCA   GTCTCATAAA       ...
Using Thermal GradientsAMPLIFICATION           • Thermal optimization is often the first parameter an individual          ...
Assay optimizationAMPLIFICATION                               For 1             Rev 1                   5’                ...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQTM SYBR® Green Supermix: 5ul Assay 95oC 60se...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Gradient analysisAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec /...
Optimal Annealing RangeAMPLIFICATION                CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 6...
Effect of Annealing Temp on C(t)AMPLIFICATION                                                            C(t) vs Annealing...
Different reagents behave very differentlyAMPLIFICATION                                                  C(t) vs Annealing...
CCL26 primer designAMPLIFICATION                CTGGAATTGA   GGCTGAGCCA   AAGACCCCAG        GGCCGTCTCA   GTCTCATAAA       ...
How did they fare?AMPLIFICATION            CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50...
CCL26 primer designAMPLIFICATION                CTGGAATTGA   GGCTGAGCCA   AAGACCCCAG        GGCCGTCTCA   GTCTCATAAA       ...
Primer TitrationAMPLIFICATION           • Primer concentration plays an important role in qPCR             amplification. ...
100nM each PrimerAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay   98oC 30sec /...
100nM each PrimerAMPLIFICATION                                                                                        Repl...
200nM each PrimerAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay   98oC 30sec /...
200nM each PrimerAMPLIFICATION                                                                                        Repl...
300nM each PrimerAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay   98oC 30sec /...
300nM each PrimerAMPLIFICATION                                                                                        Repl...
400nM each PrimerAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay   98oC 30sec /...
400nM each PrimerAMPLIFICATION                                                                                        Repl...
600nM each PrimerAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay   98oC 30sec /...
600nM each PrimerAMPLIFICATION                                                                                        Repl...
800nM each PrimerAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay   98oC 30sec /...
800nM each PrimerAMPLIFICATION                                                                                        Repl...
Melt curveAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay   98oC 30sec / 50x 95...
Increased primer concentrations can increase                                                                        nonspe...
Large dynamic rangeAMPLIFICATION           GAPDH amplified using Bio-Rad SsoFast EVAGreen Supermix: 20ul Assay   98oC 30se...
High sensitivity assayAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay   98oC 30...
High sensitivity assayAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay   98oC 30...
Standard CurveAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay   98oC 30sec / 50...
2nd Structures on templateAMPLIFICATION                CTGGAATTGA   GGCTGAGCCA       AAGACCCCAG      GGCCGTCTCA    GTCTCAT...
200nM forward -- 100nM reverseAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay  ...
200nM forward -- 100nM reverseAMPLIFICATION                                                                               ...
200nM forward -- 200nM reverseAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay  ...
200nM forward -- 200nM reverseAMPLIFICATION                                                                               ...
200nM forward -- 300nM reverseAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay  ...
200nM forward -- 300nM reverseAMPLIFICATION                                                                               ...
200nM forward -- 400nM reverseAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay  ...
200nM forward -- 400nM reverseAMPLIFICATION                                                                               ...
200nM forward -- 600nM reverseAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay  ...
200nM forward -- 600nM reverseAMPLIFICATION                                                                               ...
200nM forward -- 800nM reverseAMPLIFICATION           CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay  ...
200nM forward -- 800nM reverseAMPLIFICATION                                                                               ...
AT rich sequences on templateAMPLIFICATION                CTGGAATTGA   GGCTGAGCCA       AAGACCCCAG      GGCCGTCTCA    GTCT...
Large ampliconsAMPLIFICATION           • Classic qPCR rules dictate that amplification products be             between 75 ...
Large amplicons – dynamic rangeAMPLIFICATION                                                         •B-Actin 1076 bp ampl...
Large amplicons - sensitivityAMPLIFICATION                                                          •B-Actin 1076 bp ampli...
Sequence HomologyAMPLIFICATION           • Designing primers on a region of template sequence             homologous to an...
InhibitorsAMPLIFICATION                Blood Serum                                                                        ...
InhibitorsAMPLIFICATION     Blood Serum                                                                <0.0098 %          ...
Scale effect on reproducibilityAMPLIFICATION                                   45                                         ...
Quality reagents are scalableAMPLIFICATION                           45                                                   ...
ThroughputAMPLIFICATION                               •   The CFX384 real-time PCR                                   detec...
SpeedAMPLIFICATION                                   SsoFast EvaGreen Supermix                                   Sso7d fro...
ConclusionsAMPLIFICATION           • The key to speeding up any screening process begins with             proper design an...
AMPLIFICATION           • Thank You!                          www.bio-rad.com/genomics/pcrsupport
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Fast qPCR assay optimization and validation techniques for HTS

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Learn how to perform fast qPCR for high throughput screening.

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Fast qPCR assay optimization and validation techniques for HTS

  1. 1. Fast qPCR assayoptimization and validation techniques for HTS Francisco Bizouarn International Field Application Specialist Gene Expression Division Bio-Rad Laboratories
  2. 2. Generating a good assay is easyAMPLIFICATION • Following a few simple steps: – Design assay – Run a gradient – Run a dilution series to validate assay dynamic range • A little extra effort in the beginning will make a tremendous amount of difference in the analysis when the assay is run hundreds or thousands of times. www.bio-rad.com/genomics/pcrsupport
  3. 3. Assay designAMPLIFICATION • Often oversimplified by the use of software or by many companies that offer design services and softwares. • Design a critical parameter. • Following a few simple steps will increase the chances of designing a successful assay. • Let’s use an example: target CCL26 in HUVEC cells www.bio-rad.com/genomics/pcrsupport
  4. 4. CCL26 cDNA sequenceAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT www.bio-rad.com/genomics/pcrsupport
  5. 5. Sequence Alignment (BLAST)AMPLIFICATION • Prior to designing primers, it’s a good idea to run a sequence homology analysis. (BLAST) • This allows the identification of sequences that may co- amplify or interfere with our intended target. • The data is freely available, so why not make use of it. • http://blast.ncbi.nlm.nih.gov www.bio-rad.com/genomics/pcrsupport
  6. 6. CCL26 with homologous sequencesAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT www.bio-rad.com/genomics/pcrsupport
  7. 7. CCL26 with homologous sequencesAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT www.bio-rad.com/genomics/pcrsupport
  8. 8. 2nd structure analysis of CCL26AMPLIFICATION • DNA is often seen as a linear polymer. • In it’s single stranded state (cDNA) regions that have complimentary sequences will tend to hybridize generating hairpins that may inhibit primer annealing. • Avoiding these sequences when possible will improve amplification effiecency. • http://mfold.bioinfo.rpi.edu/cgi-bin/dna- form1.cgi www.bio-rad.com/genomics/pcrsupport
  9. 9. CCL26 with 2nd structuresAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT www.bio-rad.com/genomics/pcrsupport
  10. 10. CCL26 with 2nd structuresAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT www.bio-rad.com/genomics/pcrsupport
  11. 11. Amplicon sizeAMPLIFICATION • Classic qPCR rules dictate that amplification products be between 75 and 200 bp in length. • These limits are not absolute. It is better to design a larger amplicon than to risk target specificity and primer annealing issues • New “ultra fast” reagents allow much larger amplicons to be used in qPCR. www.bio-rad.com/genomics/pcrsupport
  12. 12. Design primersAMPLIFICATION • Some primer design packages will take both sequence homology and secondary structure issues into account when designing assays. • Due to the restrictions imposed on the design software, they can fail. • Although not recommended, designing assays by “thumb” can be performed. GCGGAATCTT TTCTGAAGGC TACATGGACC • There are also databases of freely available primers and probes that have been previously tested. www.bio-rad.com/genomics/pcrsupport
  13. 13. CCL26 primer designAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT www.bio-rad.com/genomics/pcrsupport
  14. 14. Using Thermal GradientsAMPLIFICATION • Thermal optimization is often the first parameter an individual using PCR will test to get the optimal reaction conditions. • Unfortunately many qPCR users often ignore this parameter, as though antiquated, in favor of more elaborate primer design software packages. • Finding the correct annealing temperature at which to run an assay is critical. www.bio-rad.com/genomics/pcrsupport
  15. 15. Assay optimizationAMPLIFICATION For 1 Rev 1 5’ 3’ For 2 Rev 2 For 1 For 2 Rev 1 Rev 2 10o above design { 5o below design www.bio-rad.com/genomics/pcrsupport
  16. 16. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQTM SYBR® Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  17. 17. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  18. 18. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  19. 19. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  20. 20. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  21. 21. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  22. 22. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  23. 23. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  24. 24. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  25. 25. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  26. 26. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  27. 27. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  28. 28. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  29. 29. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  30. 30. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  31. 31. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  32. 32. Gradient analysisAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  33. 33. Optimal Annealing RangeAMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  34. 34. Effect of Annealing Temp on C(t)AMPLIFICATION C(t) vs Annealing Temp 72 70 68 66 Annealing Temp 64 62 60 58 56 54 52 25 30 35 40 45 50 55 C(t) CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  35. 35. Different reagents behave very differentlyAMPLIFICATION C(t) vs Annealing Temp C(t) vs Annealing Temp 72 72 70 70 68 68 66 66 Annealing Temp Annealing Temp 64 64 62 62 60 60 58 58 56 56 54 54 52 52 25 30 35 40 45 50 55 25 30 35 40 45 50 55 C(t) C(t) CCl26 amplified using Bio-Rad Sso Fast EVA Green Supermix: CCl26 amplified using Other Reagent A: 5ul Assay 5ul Assay98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt 95oC 5min / 50x 95oC 15 sec 55-70oC 60 sec / melt analysis analysis C(t) vs Annealing Temp C(t) vs Annealing Temp 72 72 70 70 68 68 66 66 Annealing Temp Annealing Temp 64 64 62 62 60 60 58 58 56 56 54 54 52 52 25 30 35 40 45 50 55 25 30 35 40 45 50 55 C(t) C(t) CCl26 amplified using Other Reagent B: 5 ul Assay CCl26 amplified using Other Reagent C: 5ul Assay 95oC 20sec / 50x 95oC 3 sec 55-70oC 30 sec / melt analysis 95oC 20sec / 50x 95oC 3 sec 55-70oC 30 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  36. 36. CCL26 primer designAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT www.bio-rad.com/genomics/pcrsupport
  37. 37. How did they fare?AMPLIFICATION CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  38. 38. CCL26 primer designAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT www.bio-rad.com/genomics/pcrsupport
  39. 39. Primer TitrationAMPLIFICATION • Primer concentration plays an important role in qPCR amplification. • Typical concentrations go from 200nM to 500nM but can vary from 50nM to 800nM and sometimes higher. • High primer concentrations dramatically increase the incidence of non specific amplification and primer-dimers. • Reasonably well designed assays work best at normal primer concentrations www.bio-rad.com/genomics/pcrsupport
  40. 40. 100nM each PrimerAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  41. 41. 100nM each PrimerAMPLIFICATION Replicates Mean C(t) : 27.24 Standard Deviation : 0.284 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  42. 42. 200nM each PrimerAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  43. 43. 200nM each PrimerAMPLIFICATION Replicates Mean C(t) : 26.59 Standard Deviation : 0.184 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  44. 44. 300nM each PrimerAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  45. 45. 300nM each PrimerAMPLIFICATION Replicates Mean C(t) : 26.54 Standard Deviation : 0.185 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  46. 46. 400nM each PrimerAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  47. 47. 400nM each PrimerAMPLIFICATION Replicates Mean C(t) : 26.51 Standard Deviation : 0.269 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  48. 48. 600nM each PrimerAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  49. 49. 600nM each PrimerAMPLIFICATION Replicates Mean C(t) : 26.49 Standard Deviation : 0.233 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  50. 50. 800nM each PrimerAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  51. 51. 800nM each PrimerAMPLIFICATION Replicates Mean C(t) : 26.58 Standard Deviation : 0.193 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  52. 52. Melt curveAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  53. 53. Increased primer concentrations can increase nonspecific amplification products generated.AMPLIFICATION 200nM Socs1 primers Conditions: 95oC 3min / 50x 95oC 10 sec 55-70oC 30 sec / melt analysis 400nM Socs1 primers Conditions: 95oC 3min / 50x 95oC 10 sec 55-70oC 30 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  54. 54. Large dynamic rangeAMPLIFICATION GAPDH amplified using Bio-Rad SsoFast EVAGreen Supermix: 20ul Assay 98oC 30sec / 50x 95oC 1 sec 60oC 1 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  55. 55. High sensitivity assayAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 58oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  56. 56. High sensitivity assayAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 58oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  57. 57. Standard CurveAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 58oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  58. 58. 2nd Structures on templateAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT Maintain forward primer at 200nM Titer reverse primer www.bio-rad.com/genomics/pcrsupport
  59. 59. 200nM forward -- 100nM reverseAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  60. 60. 200nM forward -- 100nM reverseAMPLIFICATION Replicates Mean C(t) : 35.91 Standard Deviation : 0.540 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  61. 61. 200nM forward -- 200nM reverseAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  62. 62. 200nM forward -- 200nM reverseAMPLIFICATION Replicates Mean C(t) : 31.13 Standard Deviation : 0.200 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  63. 63. 200nM forward -- 300nM reverseAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  64. 64. 200nM forward -- 300nM reverseAMPLIFICATION Replicates Mean C(t) : 29.33 Standard Deviation : 0.209 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  65. 65. 200nM forward -- 400nM reverseAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  66. 66. 200nM forward -- 400nM reverseAMPLIFICATION Replicates Mean C(t) : 28.20 Standard Deviation : 0.168 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  67. 67. 200nM forward -- 600nM reverseAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  68. 68. 200nM forward -- 600nM reverseAMPLIFICATION Replicates Mean C(t) : 27.19 Standard Deviation : 0.104 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  69. 69. 200nM forward -- 800nM reverseAMPLIFICATION CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  70. 70. 200nM forward -- 800nM reverseAMPLIFICATION Replicates Mean C(t) : 26.95 Standard Deviation : 0.062 CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  71. 71. AT rich sequences on templateAMPLIFICATION CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT TGACTTGTTC AT Maintain forward primer at 200nM Titer reverse primer www.bio-rad.com/genomics/pcrsupport
  72. 72. Large ampliconsAMPLIFICATION • Classic qPCR rules dictate that amplification products be between 75 and 200 bp in length. • New “ultra fast” reagents allow much larger amplicons to be used in qPCR. • Extending the size of the amplicon should be considered when trying to circumvent secondary structures, sequence homology and unfavorable regions. • Proper validation is required. www.bio-rad.com/genomics/pcrsupport
  73. 73. Large amplicons – dynamic rangeAMPLIFICATION •B-Actin 1076 bp amplicon from plasmid •109 to 10 copy per well 10 fold dilution 109 copies series •5 ul asay run on CFX384 using Bio- Rad’s SsoFast EVA Green Supermix 10 copies •Protocol : 98oC 3 min 45 x 95oC 1 sec 66oC 5 sec melt curve www.bio-rad.com/genomics/pcrsupport
  74. 74. Large amplicons - sensitivityAMPLIFICATION •B-Actin 1076 bp amplicon from plasmid •105 to 200 copy per well 2 fold dilution series 105 copies •5 ul asay run on CFX384 using Bio- Rad’s SsoFast EVA Green Supermix 200 copies •Protocol : 98oC 3 min 45 x 95oC 1 sec 66oC 5 sec melt curve www.bio-rad.com/genomics/pcrsupport
  75. 75. Sequence HomologyAMPLIFICATION • Designing primers on a region of template sequence homologous to another gene should be avoided if possible. • When inevitable, a single primer can be designed to anneal on a homologous region for a series of genes. The other primer should annealing on a clean region or one that has no homology with genes annealed by the first primer. • Multiple primers should be designed and tested. • If a single primer anneals multiple targets, it will generate a linear amplification of DNA where as if both primers anneal, the amplification will be exponential. www.bio-rad.com/genomics/pcrsupport
  76. 76. InhibitorsAMPLIFICATION Blood Serum <2.5 % 10 % CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x95oC 1 sec 60oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  77. 77. InhibitorsAMPLIFICATION Blood Serum <0.0098 % 0.039 % <0.0089% 0.039% CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: CCl26 amplified using Other Reagent A: 5ul Assay 5ul Assay 95oC 3 min / 50x 95oC 10 sec 60oC 60 sec / melt 95oC 5min / 50x 95oC 15 sec 60oC 60 sec / melt analysis <0.0089% <0.0089% 0.039% 0.039% CCl26 amplified using Other Reagent B: 5ul Assay CCl26 amplified using Other Reagent C: 5ul Assay 95oC 20sec / 50x 95oC 3 sec 60oC 30 sec / melt analysis 95oC 20sec / 50x 95oC 3 sec 60oC 30 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  78. 78. Scale effect on reproducibilityAMPLIFICATION 45 6 40 5 35 4 delta C(t) 40 pg X C(t) 30 1 pg X 3 delta 25 2 20 1 15 0 2.5 5 10 20 Volume (ul) CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 2.5-20 ul Assay 98oC 30sec / 50x 95oC 1 sec 60oC 5 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  79. 79. Quality reagents are scalableAMPLIFICATION 45 45 6 6 40 40 5 5 35 35 4 4 delta C(t) 40 pg 40 pg delta C(t) C(t) C(t) 30 1 pg 30 1 pg 3 3 delta delta 25 25 2 2 20 1 20 1 15 0 15 0 2.5 5 10 20 2.5 5 10 20 Volume (ul) Volume (ul) CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: CCl26 amplified using Other Reagent A: 2.5-20 ul Assay 2.5-20 ul Assay 95oC 3 min / 50x 95oC 10 sec 60oC 60 sec / melt 95oC 5min / 50x 95oC 15 sec 60oC 60 sec / melt analysis 45 45 6 6 40 40 5 5 35 35 4 4 delta C(t) delta C(t) 40 pg 40 pg C(t) C(t) 30 1 pg 30 1 pg 3 3 delta delta 25 25 2 2 20 1 20 1 15 0 15 0 2.5 5 10 20 2.5 5 10 20 Volume (ul) Volume (ul) CCl26 amplified using Other Reagent B: 2.5-20 ul Assay CCl26 amplified using Other Reagent C: 2.5-20 ul Assay 95oC 20sec / 50x 95oC 3 sec 60oC 30 sec / melt analysis 95oC 20sec / 50x 95oC 3 sec 60oC 30 sec / melt analysis www.bio-rad.com/genomics/pcrsupport
  80. 80. ThroughputAMPLIFICATION • The CFX384 real-time PCR detection system brings flexibility and ease of use to researchers performing high-throughput real- time PCR in a 384-well format. • With up to 4-target detection, unsurpassed thermal cycler performance, and powerful, yet easy-to-use software, the CFX384 system has been designed for the way you work. – FAST – shorten the time from experiment setup to results – FRIENDLY – a new standard for ease of use, delivering data you can trust with no maintenance – FLEXIBLE – customize a set up that fits individual laboratory needs www.bio-rad.com/genomics/pcrsupport
  81. 81. SpeedAMPLIFICATION SsoFast EvaGreen Supermix Sso7d from Sulfolobus solfataricus – 7kD, 63 aa. – Thermostable (Tm >90°C) – No sequence preference – Binds to dsDNA (3-6 bp/protein molecule) – Monomeric • Minimal inhibition of PCR by use of EvaGreen • Higher activity • Tolerant to PCR inhibitors www.bio-rad.com/genomics/pcrsupport
  82. 82. ConclusionsAMPLIFICATION • The key to speeding up any screening process begins with proper design and optimization. • qPCR assay optimization and dynamic range validation require very little time and effort and help guarantee that the results will be reproducible and comparable form experiment to experiment. • If potentially interfering elements are discovered at the design and optimization phases, they can be accounted for and possible corrected. • As demands for shorter run times increase, proper care in the selection of reagents and instruments is required. www.bio-rad.com/genomics/pcrsupport
  83. 83. AMPLIFICATION • Thank You! www.bio-rad.com/genomics/pcrsupport

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