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Optimice y seleccione la mejor polimerasa para sus reacciones de PCR habituales
1-Introducción a la PCR 2-Optimización de una PCR 3-Surecycler 8800 software 4-Overview de polimerasas 5-Revisión de RT-PCR
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Introducción a la PCR
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Se fundamenta en la propiedad natural de las ADN polimerasas para replicar hebras de ADN, para lo cual emplea ciclos de altas y bajas temperaturas alternadas para separar las hebras de ADN recién formadas entre sí tras cada fase de replicación y, a continuación, dejar que vuelvan a unirse a polimerasas para que vuelvan a duplicarlas
Las temperaturas del ciclo (95 °C en las fases de desnaturalización del ADN) suponen la inmediata desnaturalización de toda proteína,,
Polimerasas de m.o
Thermus aquaticus (polimerasaTaq) Pyrococcus furiosus (Pfu) Thermococcus litoralis (Vent) Thermus termophilus (Tth).
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Introducción a la PCR
Los 7 componentes de una PCR: 1-Los 4 desoxirribonucleósidos-trifosfato (dNTP), sustratos para polimerizar nuevo ADN. 2-Dos cebadores o iniciadores (primers), oligonucleótidos que son, cada uno, complementarios a una de las dos hebras del ADN. Son secuencias cortas, de entre 6 y 40 nucleotidos. Deben estar situados enfrentados y a no mucha distancia ,definen los extremos de la secuencia que se desea replicar.
- 3-Se suele usar magnesio (Mg2+) Cofactores de la polimerasa. 4-Una solución tampón (buffer) que mantiene el pH adecuado para el funcionamiento de la ADN polimerasa. 5-ADN polimerasa 6- ADN molde, que contiene la región de ADN que se va a amplificar. 7-Termociclador, el aparato que va a mantener la temperatura necesaria en cada una de las etapas que conforman un ciclo. http://genomics.agilent.com/
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PCR; 20 a 35 cambios repetidos de temperatura llamados ciclos; cada ciclo suele consistir en 3 pasos a diferentes temperaturas.
Inicio 94-96 °C 1-9 minutos Desnaturalización(94-95 °C) 60 seg La temperatura depende: de la proporción de G+C y longitud de la hebra 95ºC standard y 92ºC para grandes hebras) Alineamiento40-68 °C durante 20-40 segundos El cebador se unirá a su secuencia complementaria en el ADN molde.. Los puentes de hidrógeno estables entre las cadenas de ADN (unión ADN-ADN) sólo se forman cuando la secuencia del cebador es muy similar a la secuencia del ADN molde.. Los cebadores actuarán como límites de la región de la molécula que va a ser amplificada. Elongación El tiempo de extensión depende + ADN polimerasa +Longitud del fragmento de ADN que se va a amplificar. **A la temperatura óptima la polimerasa de ADN polimerizará mil bases en un minuto…… polimerasa Taq 75-80 °C Elongación final 70-74 °C 5-15 minutos tras el último ciclo de PCR.
-Electroforesis en gel de agarosa……fragmentos grandes -Gel de acrilamida……………………...fragmentos pequeños *Marcador de peso molecular
Introducción a la PCR
Stratagene PCR Enzymes
2º cycle
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PCR Optimización
PH -Los buffer contienen agentes reguladores del pH -El pH ideal es de 8.4 -El pH del Tris buffer disminuye a alta temperaturas. Concentración de sales Los buffers contienen 50mM(taq) o 10 mM(Pfu) KCl para proporcionar la suficiente fuerza iónica. Magnesium Los buffers frecuentemente contienen Mg 2+ como cofactor para la actividad de la enzima. Taq polimerasa es más sensible al Mg que la Pfu en la concentración óptima dNTP -La concentración óptima suele estar entorno a 1mM. -Un exceso de la concentración de dNTP reduce la precisión de la polimerasa y requiere de más Mg2+ en la reacción. Template La cantidad mínima suele rondar de 10 a 1000 copias por reacción, aproximadamente 5-100ng de template. El ratio A260/A280 debería estar entre 1.8 a 2.0. La cantidad recomendada 25-100ng en 100ul de reacción en mayoría de las veces* Para alto contenido en GC se ha de añadir entre 1% a 10% DMSO para reducir estructuras secundarias. Glicerol entre el 5-10%.
Aditivos 1-Glicerol(5-10%),Formamida(1-5%) o DMSO (2-10%) puede ser añadido en casos de moldes de ADN con alto GC. 2-Betaína(0,5M-2M) es útil para alto GC pero no para nuestras Pfu. (Produce una tritación a una concentración optima) 3-BSA (por encima de 0.8ug/ul)
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PCR Optimización y diseño Optimiza la concentración -La concentración optima suele ser entre 100-500nM. Un aumento excesivo puede inhibir la reacción. -Conversores habituales; 330g mol * bp = peso molecular de ssDNA 660g mol*bp= peso molecular de dsDNA *Un ejemplo: ¿Cómo puedo conseguir 10mM de solución empezando con 24 nmol de oligo? 1- Convertir los 10mM a 10 mmol/ml. 2- Cálcula el volumen que necesitas resuspender tus oligos en función de tu concentración inicial. 10mmol/ml= 24 nmol/X ml, Luego X= 2,4 ml
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Diseño de Primers Cada cebador debe integrar entre 18 y 24 bases. Los cebadores muy cortos hacen que nuestra PCR no sea muy específica, y los que se excedan en longitud harán que perdamos rendimiento en la reacción. -La proporción entre bases púricas y pirimidínicas sea 1:1 (40-60% a lo sumo), y que empiecen y terminen con 1 ó 2 bases púricas. -Los cebadores no deben incluir en su secuencia regiones que sean complementarias entre sí, o se formarán dímeros entre ellos. http://eu.idtdna.com/Home/Home.aspx -Intentar terminaciones en el 3´ en bases GC para facilitar el proceso de anillamiento. -Evitar secuencias invertidas para prevenir formación de estructuras secundarias, la cuales pueden facilitar eliminar hibridaciones en el template.
-La temperatura de anillamiento en la PCR debería ser entre 5ºC a 10ºC por debajo de la temperatura de melting (Tm). Tm (ºC)= 2(#A + #T) + 4(#G + #C) -La Tm óptima suele estar entre 55ºC y 80ºC para mejorar el rendimiento
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http://www.biocompare.com/Multimedia/Video/764/Watch-Video-Agilent-SureCycler-8800-%E2%80%9CAll-in-one%E2%80%9D-Thermal-Cycler.html
Surecycler software
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1 2
3 4
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5
1**
6
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4-Overview de Polimerasas
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Attributes of PCR Enzymes
Fidelity (Amplicons with the correct sequence)
Robustness (% of reactions that successfully amplify)
Yield downstream aplications required larger quantities of DNA
Sensitivity (Low amounts of starting material)
Specificity (Amplification of the correct target)
Speed (High throughput labs and individual protocols)
Target length mainly GC-rich templates
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PCR Enzyme Product Positioning
Agilent’s Stratagene Division - Key DNA Polymerases Key Attributes Product
Highest Fidelity PfuUltra II Fusion HS
Highest Yield
Most Robust
Lowest cost for High Fidelity PCR
Herculase II Fusion*
Highest Sensitivity PicoMaxx
TA/UA Cloning Easy-A
Lowest cost for routine PCR (HS) Paq5000 HS DNA Polymerase
Incorporation of dUTP PfuTurbo Cx
*Length (up to 23kb) – Herculase II with optimized buffer and cycling conditions
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ArchaeMaxx Polymerase-Enhancing Factor
Accumulation of dUTP in reaction can stop extension by archael polymerases (PCR poisoning)
ArchaeMaxx is Archael dUTPase
• Don´t have DNA polymerase and exonuclease activities Overcomes poisoning in extreme PCR conditions
The longer the overall PCR, the more dUTP can accumulate Complex/challenging and long templates
Results in enhanced overall PCR performance
Higher yields Greater target length capability
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Value: PfuUltra II HS Fusion DNA Polymerase • The most accurate amplification in our industry
• 1 error every 2,500,000 bp
• Peer reviewed, published results in major journals
• Extreme speed leading to reduced overall run time • 15 sec/kb extension
• Extended length capability • 19 kb with gDNA
• Contains ArchaeMaxx Factor to prevent poisoning by dUTP • Highly specific – only sold as hot-start formulation • Convenience with master mix options • No impact on performance when left at room temperature for 24 hours
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Pfu-Based Fusion Protein
• Fusion enzymes are a combination of a high affinity double-stranded DNA binding domain with a Pfu-based DNA polymerase
• Serves to better anchor the DNA polymerase preventing early dissociation from the DNA template
• Improved processivity for the incorporation of more nucleotides per binding event • This enhances PCR yields and shortens extension times
DNA binding protein Pfu-based DNA polymerase
N C Proofreading Polymerization
PfuUltra II :Quick Time-to-Results
PfuUltra II
Common proof reader
Common proof reader
Common proof reader
Taq
Taq
Taq
2.6kb
6kb
12kb
Total Cycling Time
52 minutes
2 hours 30 minutes
2 hours 30 minutes
1 hour 17 minutes
6 hours 52 minutes Failed amplification
Failed amplification
2 hours 40 minutes 13 hours 44 minutes
PfuUltra II
PfuUltra II
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Error Rate, Accuracy… Who Cares?
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1-kb amplicon 5-kb amplicon 10-kb amplicon
Pfu, PfuTurbo, Herculase II1,2
(Agilent) 1.3 ± 0.2 770,000 2.6 13 26PfuUltra/PfuUltra II(Agilent) 0.4 ± 0.04 2,500,000 0.8 4 8Tgo(Roche) 2.2 ± 0.1 450,000 4.4 N.R. N.R.DeepVent1
(New England Biolabs) 2.7 ± 0.2 370,000 5.4 N.R. N.R.Vent1
(New England Biolabs) 2.8 ± 0.9 360,000 5.6 N.R. N.R.Platinum Pfx4
(Life Technologies) (KOD1) 3.5 ± 1.0 290,000 7 35 70
Herculase3
(Agilent)(Maj Pfu + Min Taq ) 2.8 ± 0.5 360,000 5.6 28 56Platinum Taq High Fidelity3
(Life Technologies)(Maj Taq + Min DeepVent™) 5.8 ± 0.3 170,000 11.6 58 100Advantage-HF3
(CloneTech)(Maj KlenTaq + Min Proofreading DNA Pol) 6.1 ± 0.07 160,000 12.2 N.R. N.R.
Taq 1
(Multiple suppliers) 8.0 ± 3.9 125,000 16 80 N.R.9.1 ± 2.4 110,000 18.2 91 N.R.
N.R.: Not recommended for 5- to 10-kb target sizes
References:
Proofreading DNA Polymerases
High-fidelity blends
No Proofreading
Percentage of clones with mutations(10⁶-fold amplification)DNA Polymerase Error Ratea
(x10ˉ⁶ ± S.D.)
Accuracy(error rate⁻¹ in bases)
aError rates were measured in each enzyme's recommended PCR buffer using the cycling conditions described in Cline et al. (1996). PCRs (50µl) contained 0.2µм each primer, 205ng or 200µм each nucleotide, 2.5ng of target DNA, and 2.5 units of DNA polymerase, with the following manufacturer-recommended exceptions: Platinum Pfx: 300 µм each nucleotide, 1.25 units of enzyme, and 68°C extension temperature; Tgo : 1 unit of enzyme and 0.4 µм each primer, Vent: 1 unit of enzyme.
1Cline J., Braman J.C., and Hogrefe H.H. 1996, PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 24: 3546-3551.
2Hogrefe H.H., Scott B., Nielson K., Hedden V., Hansen C., Cline J., Bal F., Amberg J., Allen R., and Madden M. 1997. Novel PCR enhancing factor improves performance of Pfu DNA polymerase, Strategies 10: 93-96.3Borns M, and Hogrefe H. 2000a. Unique DNA polymerase formulation excels in a broad range of PCR application. Strategies 13: 1-3.4Borns M, and Hogrefe H. 2000b. Unique enhanced DNA polymerase delivers high fidelity and great PCR performance. Strategies 13: 76-79.
10/19/2011
* PCR conducted using recommended buffer and cycling conditions
PfuUltra II: Extended Target Length Capability
1 2 3 4 6 9 12 19 1 2 3 4 6 9 12 19 1 2 3 4 6 9 12 19
PfuUltra II* Phusion* Pfx50* Genomic DNA target length (kb):
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PfuUltra II Ordering Information
Description Reactions / Units Catalogue #
PfuUltra II Fusion HS DNA Polymerase 40 rxns (50uL) 600670
PfuUltra II Fusion HS DNA Polymerase
200 rxns (50uL) 600672
PfuUltra II Fusion HS DNA Polymerase
400 rxns (50uL) 600674
PfuUltra II Hotstart PCR Master Mix
100 rxns (50uL) 600850
PfuUltra II Hotstart PCR Master Mix
400 rxns (50uL) 600852
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Value: Herculase II • Superior yields • Most robust DNA polymerase
• Successfully amplify a broad range of targets including difficult and GC-rich targets
• Amplification of precious samples
• Increased speed • 30 sec/kb extension**
• Length - Up to 12kb genomic (unoptimized), 23kb genomic (optimized) • High sensitivity for amplification of as little as 1 ng of DNA • Prevent poisoning of dUTP with ArchaeMaxx Factor • Most economically priced high fidelity enzyme in the market
• 1 error in 750,000 bp
**Extension times can be adjusted up or down depending on customer’s targets and needs for yield/sensitivity
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10/19/2011
Herculase II: Higher Yields than Other High Fidelity DNA Polymerases
6kb
1.7kb
Ext. time (sec/kb): 15 30 45 60 15 30 45 60 15 30 45 60 15 30 45 60
Herculase II Phusion Pfx50 Pwo Super Yield
Ext. time (sec/kb): 15 30 45 60 15 30 45 60 15 30 45 60 15 30 45 60
Herculase II Phusion Pfx50 Pwo Super Yield
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10/19/2011
Herculase II: Reliable Amplification of GC Rich Targets
Target: IGFB Fr. X HTR MMZ5 IGFB Fr. X HTR MMZ5 IGFB Fr. X HTR MMZ5
Herculase II* Phusion/GC Buffer* AccuPrime GC*
(79%) (84%) (65%) (68%) (79%) (84%) (65%) (68%) (79%) (84%) (65%) (68%) GC content (%):
562bp 540bp 300bp 250bp
*Manufacturers’ recommendations were followed for amplifying GC-rich targets
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Superior Sensitivity with Herculase II
• 3.9-kb fragment of human α1-antitrypsin gene amplified from 1-300 ng input amount
• All reactions performed using manufacturer’s recommended buffer and protocol
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PCR Polymerase Applications Application Competitor
Polymerase Agilent Alternative
Benefit to Customer
Next-Gen Sequencing
Phusion (Finnzymes) Platinum PCR Supermix (Life Tech)
Herculase II •SureSelect - Customer does not risk support/replacement of product if experiment is not successful. Save costs of SureSelect and sequencing.
•Higher yields mean fewer cycles of amplification are required which saves the customer time but more importantly reduces bias introduced from PCR and reduces errors which occur with all DNA polymerases
•Vs Platinum PCR Supermix (not Phusion) – Customer can achieve a much higher throughput with significantly faster time to results due to fusion technology
•***SureSelect - Preservation of size distribution to reduce wasted sequencing saving costs and time to run bioinformatics
SureSelect / Herculase II Publication (March 2010): SureSelect X Chromosome kit with Herculase II and complimentary analysis by Agilent aCGH to identify mutations and deletions in the X chromosome gene PHF6 in T-cell acute lymphoblastic leukemia Nature Genetics, Adolfo Ferrando and colleagues, at Columbia University http://opengenomics.com/Papers/PHF6_mutations_in_Tcell
Comparing PfuUltra II and Herculase II Fusion DNA Polymerases
• PfuUltra II when fidelity, speed, and specificity are most important
• Herculase II when high fidelity and yield is needed on a routine basis across multiple targets
Parameter PfuUltra II Fusion HS Herculase II Fusion
Fidelity Best High ( = Pfu) Hot-start Yes No
ArchaeMaxx Yes Yes Cycling (extension) Fast (15 sec/kb) Fast (30 sec/kb)
Yield High Best
GC-rich templates Good performance Best
Length 0.1-19kb genomic 0.1-12kb genomic
Pricing Premium Economic
Stratagene University - PCR Reagents
7/13/2009 Page 31
PfuTurbo Cx
Proofreading polymerase
Only available in hotstart formulation
Robust
Completely overcomes Uracil stalling – Reads through Uracil in template or extending strand, efficient
incorporation of dUTP
Agilent provides the only commercially available high fidelity polymerase which reads through Uracil
Stratagene University - PCR Reagents
7/13/2009 Page 32
Target Customer PfuTurbo Cx
Epigenetics
Methylation
Bisulfite Sequencing
UNG decontamination protocols
Require accuracy from PCR reaction with dUTP present or Uracil in the template
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PfuTurbo Cx for Bisulfite Sequencing
• Proofreading polymerases stall at dUTP, thus impacting efficiency of amplification – PfuTurboCx is designed to read through Uracil without stalling
• Ideal polymerase for large-scale random bisulfite sequencing in genome-wide analysis of DNA methylation
Paq5000
Economic alternative to Taq • Optimal performance may require some optimization when substituted directly
into Taq protocols
Optimized to provide higher yield and better sensitivity than Taq
Reduced extension time by 50% compared to Taq • 30 sec/kb extension time
High specificity with hot-start formulation, master mix
Improved target length • Amplify up to 6kb genomic DNA
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Supporting Proof: Increased Specificity and Yield Paq5000 HS vs. Standard Taq
Amplification of a 3.9 kb fragment of the Human α1 Antitrypsin gene using Paq5000 Hotstart DNA Polymerase and standard Taq from various suppliers. Paq5000 Hotstart DNA Polymerase reactions were performed with extension times of 30 sec/kb. All other reactions were performed with extension times of 1 min/kb and using each manufacturer’s recommended buffer, enzyme concentration and cycling conditions. ( lanes 5-7, NEB Taq; lanes 8-10, Invitrogen Taq; lanes 11-13, Roche Taq; lanes 14-16, Paq5000 with no hot start).
Paq5000 HS NEB Taq Invitrogen Roche Taq Paq5000
Paq5000 Hotstart DNA Polymerase – Features/Benefits
Feature Benefit
Novel hot start capability allows room temperature set-up of PCR reactions without compromising specificity due to mispriming.
Added convenience.
Novel hot start capability reduces non-specific bands and has been shown to eliminate non-specific bands with even the most difficult primer/template combinations.
Achieve greater PCR success.
Fast cycling conditions (30 sec/kb extension time vs. I min/kb for Taq) & short 2 minute hot start.
Save time and increase throughput.
Amplify up to 6kb genomic DNA. No specialized PCR enzyme required for routine PCR on longer targets.
Priced as low as 15 cents per unit – up to 66% less than any major competitor hotstart!
Save money on routine PCR.
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Resolución de problemas
PCR Polymerase Applications Application Competitor
Polymerase Agilent Alternative
Benefit to Customer
Gene Synthesis
High Fidelity (Any competitor)
PfuUltra II •Highest accuracy gene synthesis = less repeating experiments = reduced cost of gene synthesis and greater chance of success •Faster PCR leads to higher throughput which allows customers to accomplish more from one instrument and in one day
Epigenetics (Methylation studies)
Taq (any competitor)
PfuTurbo Cx •More accurate amplification of desired sequence due to a high fidelity polymerase which efficiently incorporates dUTP
PCR Carryover Contamination Prevention (UNG Decontamination)
Taq (any competitor)
PfuTurbo Cx •More accurate amplification of desired sequence due to a high fidelity polymerase which efficiently incorporates dUTP
Bisulfite Sequencing
Taq (any competitor)
PfuTurbo Cx •More accurate amplification of desired sequence due to a high fidelity polymerase which efficiently incorporates dUTP
Amplification with Difficult/Long Targets (GC rich, Inhibitors Present)
AccuPrime GC-Rich (Life Tech) Other suppliers with GC specific products
Herculase II •Significantly higher yields with difficult targets mean less replication of work •More consistent amplification allows for greater confidence in reliability of the customer’s PCR •Faster amplification increases the customers throughput and saves the customers time
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5-Revisión de RT-PCR
Problem: • Reverse transcriptases (RTs) exhibit significantly higher error rates, introducing
errors at frequencies of 1 per 1,500 to 30,000 nucleotides during cDNA synthesis
Why is this important? RT induced errors are the major contributor of errors in RT-PCR, particularly when
using ultra-high fidelity PCR enzymes Importance: single base errors affect protein expression, folding or activity Pain to customer: time and effort spent to ID and correct errors after RT-PCR
AccuScript PfuUltra II RT-PCR
• Highest fidelity RT combined with highest fidelity PCR enzyme in the market
• Proofreading RT enables AccuScript a three- to six fold better accuracy than other RTs
• Reduces errors • Improves the yield of the correct cDNA
• Achieves up to 3X faster RT-PCR reaction times with the AccuScript PfuUltra II RT-PCR Kit
• High yields of full length cDNA up to 20kb with AccuScript Reverse Transcriptase • PfuUltra II amplifies up to 19kb
• Stringent quality control procedures ensure high yields of premium cDNA
High Fidelity RT-PCR
*Based on a 1 kb fragment amplified for 20 duplications
“” Note PfuUltra has been updated to PfuUltra II with an equivalent error rate as PfuUltra DNA Polymerase but much faster processivity
Full Length cDNA
Shown here is a successful PCR amplification of 0.61kb fragment located at 5’ end of the human nebulin gene, which is 20.8kb in length. This demonstrates complete reverse transcription of the human nebulin gene by AccuScript RT
The combination of AccuScript RT and PfuUltra II DNA polymerase is the only choice for accurate amplification of cDNA in RT-PCR.
RT
PCR
Mutation Frequency (MF)
RT-PCR
AccuScript RT PfuUltra II Enzyme 2.5% (1.6% + 0.9%)
SuperScript II RT PfuUltra II Enzyme 7.3% (6.4% + 0.9%)
SuperScript II RT Taq Polymerase 22.4% (6.4% + 16%)
AccuScript Reverse Transcriptase (RT)
AccuScript RT Portfolio
Application Product format Stratagene Solution Major advantages
Premium quality cDNA that is virtually error free for cloning, sequencing, protein expression
Reverse transcriptase AccuScript High-Fidelity Reverse Transcriptase
• Synthesize cDNA that contains 3-6 fold fewer errors than cDNA produced by any other reverse transcriptase
• RNase H deficient for high yields of full length cDNA
• Produce higher quality full length cDNA from 1 kb to 20 kb
First strand kit AccuScript High-Fidelity cDNA Synthesis Kit
• Complete first strand cDNA synthesis system
• Flexibility to select PCR enzyme
• Convenient kit format offers flexible oligo dT or random priming options
Premium quality cDNA amplification with the highest fidelity
2-Step RT-PCR AccuScript PfuUltra II RT-PCR Kit
• Highest fidelity RT-PCR using AccuScript and PfuUltra II enzymes
• Archival of precious samples
• Length
• RT up to 20.8 kb cDNA
• PCR enzyme up to 19 kb
Easy-A One-Tube RT-PCR System
Convenient one-step RT-PCR • The ONLY one-step RT-PCR system offering both TA/UA cloning and high
fidelity – Amplification of cDNA with A-overhangs – Six fold more accurate than Taq DNA Polymerase with the same cloning
efficiency • Minimizes contamination • Makes multiple-reaction setup fast and easy
• Single, optimized buffer for both RT and PCR phases of protocol to maximize performance
• Robust amplification of targets up to 5 kb
AffinityScript RT Advantages Robust cDNA synthesis across a broad range of temperatures
– 37°C to 55°C – Allows customers to use the optimal reaction temperature for their priming strategy or template sequence without having to change their RT
– Limited optimization required
High yield
– Detect low copy genes – Use of limited RNA
Length
– Synthesizes from 0.5 kb up to 20 kb full length cDNA
Reverse transcribe through secondary structure using elevated reaction temperature
Strict enzyme purity specifications ensures no contaminating RNase and exonuclease activities
Cost effective solution compared to competitors
AffinityScript RT-PCR Kit
One-Step • Products useful for
cloning and sequencing
• Simple, rapid setup of
multiple reactions
• Minimizes contamination
in a single tube reaction
Two-Step • Generates homogenous population of cDNA • Generate cDNA Libraries • Recovery of specific clones from a heterogonous population • First-strand cDNA for downstream QPCR specific for multiple genes
One Vs. Two-Step RT-PCR
Broadest Thermal Profile
Protocol
Run Times
Target Size AffinityScript One-Step SS III One-Step Qiagen One-Step
0.5kb 1 hour, 20 minutes 2 hours, 40 minutes 2 hours, 50 minutes
1.6kb 2 hours 3 hours, 15 minutes 3 hours, 30 minutes
2.8kb 2 hours, 39 minutes 3 hours, 55 minutes 4 hours, 10 minutes
4.8kb 4 hours 5 hours, 20 minutes 5 hours, 35 minutes
7.6kb 5 hours, 19 minutes 7 hours, 15 minutes 12 hours, 10 minutes
9.5kb 6 hours, 30 minutes 8 hours, 40 minutes 15 hours, 30 minutes
AffinityScript One-Step RT-PCR kit delivers significantly shorter run times
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Supporting Proof
Comparison of One-Step RT-PCR systems Targets of varying sizes were amplified with various commercially available one-step RT-PCR kits. Top panel: Agilent AffinityScript One-Step RT-PCR kit; Second panel: Invitrogen SuperScript III One-Step RT-PCR kit; Third panel: Qiagen OneStep RT-PCR Kit; Fourth panel: Qiagen OneStep RT-PCR kit with Q solution added. M1: 1kb DNA ladder and M2: Lambda/HindIII marker.
Agilent Invitrogen SuperScript III
Qiagen OneStep RT-PCR Kit Qiagen OneStep RT-PCR Kit + Q solution
ng RNA M1 1 1 1 1 10 10 10 100 100 M2
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Supporting Proof
1ul of PCR product from each RT-PCR reaction was run on the 2100 Bioanalyzer. Lanes 1, 5, 9: 433bp MacF1 from 1ng human skeletal muscle total RNA; lanes 2, 6, 10: 1.6kb GPT from 1ng mouse liver total RNA; lanes 3, 7, 11: 2.8kb Herc1 from 10ng human skeletal muscle total RNA; lanes 4, 8, 12: 4.8kb Neb-4 from 10ng human skeletal muscle total RNA
AffinityScript One-Step RT-PCR Kit
SuperScript III One-Step RT-PCR System with Platinum® Taq DNA
Polymerase Qiagen OneStep RT-PCR
Kit
One-Step RT-PCR Kit Comparisons
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One-Step RT-PCR Kit Comparisons
Supporting Proof
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Supporting Literature • Agilent Website - Product information page
• AffinityScript One-Step RT-PCR Kit Manual http://www.genomics.agilent.com/files/Manual/600188.pdf
• Technical Data Sheet (shown to the right, double click to open)
Publication Arezi, B. & Hogrefe, H. (2009) Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase
thermostability through tighter binding to template-primer. Nucl. Acids Res. 37: 473-481.
Conference poster Higher Resistance to Common RT-qPCR Inhibitors Displayed by AffinityScript RT (Shown to the right, double click to open)
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OFERTA PROMOCIONAL 2*1 (Exclusivo Asistentes)
Polimerasas (Herculasa, Pfu..) o Retrotranscriptasa (Affinity, AccurScript)*.
***********SORTEO DEL E-seminar*******
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