SuperScript™ III System für Erststrangsynthese

Zitierungen und Referenzen (7)

Invitrogen™

SuperScript™ III System für Erststrangsynthese

Optimiert für die Synthese von Erststrang-cDNA aus gereinigter Poly(A)⁺ oder Gesamt-RNA oder RT-PCR

Katalognummer	Menge
18080051	50 rxns

Katalognummer 18080051

Preis (EUR)

948,00

Menge:

50 rxns

Großbestellung oder individuelle Größe anfordern

Preis (EUR)

948,00

The SuperScript III First-Strand Synthesis System for RT-PCR is optimized to synthesize first-strand cDNA from purified poly(A)⁺ or total RNA. RNA targets from 100 bp to >12 kb can be detected with this system and the amount of starting material can vary from 1 pg to 5 μg of total RNA. SuperScript III Reverse Transcriptase is a version of M-MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability. The enzyme is used to synthesize cDNA at a temperature range of 42–55°C, providing increased specificity, higher yields of cDNA, and more full-length product than other reverse transcriptases. Because SuperScript III RT is not significantly inhibited by ribosomal and transfer RNA, it may be used to synthesize first-strand cDNA from a total RNA preparation.

Das SuperScript™ III Erststrang-Synthesesystem für RT-PCR ist für die Synthese von Erststrang-cDNA aus gereinigter Poly(A)⁺- oder Gesamt-RNA optimiert. Mit diesem System können RNA-Ziele von 100 bp bis > 12 kb nachgewiesen werden. Die Menge des Ausgangsmaterials kann im Bereich von 1 pg–5 µg der Gesamt-RNA liegen. SuperScript™ III Reverse Transkriptase ist eine Version von M-MLV RT, die zur Reduzierung der RNase-H-Aktivität entwickelt wurde und eine höhere thermische Stabilität bietet. Das Enzym wird zur Synthetisierung von cDNA in einem Temperaturbereich von 42–55 °C verwendet und bietet eine erhöhte Spezifität, eine höhere cDNA-Ausbeute und mehr Produkte voller Länge als andere reverse Transkriptasen. Da Superscript™ III RT nicht signifikant durch ribosomale und Transfer-RNA gehemmt wird, kann es zur Synthese von Erststrang-cDNA aus einer Gesamt-RNA-Vorbereitung verwendet werden.

Verwendung des Superscript™ III Erststrangsystems
Die cDNA-Synthese erfolgt im ersten Schritt entweder mit der Gesamt-RNA oder mit Poly(A)⁺-selektierter RNA, die an Oligo(dT), zufällige Primern oder einen genspezifischen Primer gebunden haben. Im zweiten Schritt wird die PCR in einem separaten Röhrchen mit den für das gewünschte Gen spezifischen Primern durchgeführt. Für die PCR-Reaktion empfehlen wir den Einsatz einer der folgenden DNA-Polymerasen: Platinum™ Taq DNA-Polymerase schafft automatische Hot-Start-Bedingungen mit höherer Spezifität bis zu 4 kb, Platinum™ Taq DNA-Polymerase bietet eine höhere Ausbeute und höchste Genauigkeit für Zielsequenzen bis zu 15 kb, und Platinum™ PfxDNA-Polymerase sorgt für höchste Genauigkeit für Zielsequenzen bis 12 kb.

Nur für Forschungszwecke. Nicht zur Verwendung bei diagnostischen Verfahren.

Specifications

EndprodukttypErststrang-cDNA

FormatKit

Anzahl Reaktionen50 Reaktionen

Optimale Reaktionstemperatur50 °C

Menge50 rxns

ReaktionsformatSeparate Komponenten

ReagenztypReverse Transkription

Reverse TranskriptaseSuperScript III

VersandbedingungTrockeneis

AusgangsmaterialRNA

VerfahrenReverse Transkription

Zur Verwendung mit (Anwendung)Real-Time PCR (qPCR), RT-PCR

GC-Rich PCR PerformanceHoch

Reaktionsgeschwindigkeit50 min

Unit SizeEach

Inhalt und Lagerung

Das SuperScript™ III Erststrang-Synthesesystem für RT-PCR wird geliefert mit:
• Oligo(dT)₂₀ (50 μM), 50 μl
• Zufällige Hexamere (50 ng/μl), 250 μl
• 10x RT-Puffer, 1 ml
• 0,1 M DTT, 250 μl
• 25 mM Magnesiumchlorid, 500 μl
• 10 mM dNTP-Mix, 250 μl
• SuperScript III RT (200 E/μl), 50 μl
• RNaseOUT (40 E/μl), 100 μl
• E. coli RNase H (2 E/μl), 50 μl
• DEPC-behandeltes Wasser, 1,2 ml
• HeLa-Gesamt-RNA (10 ng/μl), 20 μl
• Sense Kontroll-Primer (10 μM), 25 μl
• Antisense Kontroll-Primer (10 μM), 25 μl

Ausreichend für 50 Reaktionen, auf Basis eines Reaktionsvolumens von 20 μl. Bei -20°C lagern.

Häufig gestellte Fragen (FAQ)

I am interested in generating cDNA from total RNA. What is the difference between SuperScript III Reverse Transcriptase and SuperScript III First Strand Synthesis System for RT-PCR?

SuperScript III Reverse Transcriptase (Cat. Nos. 18080093, 18080044, 18080085) contains the stand-alone enzyme and a vial each of 5X first-strand buffer and 100 mM DTT.

SuperScript III First Strand Synthesis System for RT-PCR is a complete kit that provides the SuperScript III Reverse Transcriptase and all the other components required for synthesis of first-strand cDNA from total or poly(A)- RNA. It includes:
- Superscript III Reverse Transcriptase
- Oligo (dT)20 Primer
- Random hexamers
- 10X RT buffer
- 25 mM MgCl2
- 0.1 M DTT
- 10 mM dNTP Mix
- RNAseOUT Recombinant Ribonuclease Inhibitor
- E. coli RNAse H
- DEPC-treated water
- Total HeLa RNA control
- Sense control primer
- Anti-sense control primer
Note: The kit does not include the PCR amplification enzyme.

How can I remove genomic DNA contamination from my sample prior to performing RT-PCR?

We recommend using ezDNase (Cat. No. 11766051). ezDNase Enzyme's high specificity for double-stranded DNA enables efficient and fast genomic DNA removal without reduction in the quality or quantity of RNA. ezDNase Enzyme is heat-labile and so can be easily deactivated by heat treatment at moderate temperature (55 degrees C). These features make ezDNase Enzyme an excellent choice for genomic DNA removal prior to reverse transcription reactions.

How much RNA should be employed for first-strand cDNA synthesis?

The amount of RNA template for a cDNA synthesis is highly flexible and depends upon the amount of sample available and an individual's need. In general, 1 µg total RNA is used in a typical 20-µL RT reaction.

Find additional tips, troubleshooting help, and resources within ourReverse Transcription and RACE Support Center.

Should I treat the cDNA with RNase H prior to downstream processing?

RNase H treatment is not always necessary. Many PCR reactions work without it. However, for cDNA synthesized with RNase H-deficient reverse transcriptases (like SuperScript II, III, and IV), RNA/cDNA hybrids—especially GC-rich ones—may not denature well, reducing PCR sensitivity. RNase H treatment can help in such cases. Additionally, RNase H treatment is beneficial for cloning larger fragments.

What percentage of RNA is converted to cDNA when performing reverse transcription?

This depends highly on the quality of the sample. mRNA itself makes up 1-5% of total RNA. Depending on the primer and enzyme used, reverse transcription can covert >70% of that into cDNA.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

View all SuperScript™ III System für Erststrangsynthese FAQs

Zitierungen und Referenzen (7)

Zitierungen und Referenzen

Abstract

The UL41 protein of herpes simplex virus 1 degrades RNA by endonucleolytic cleavage in absence of other cellular or viral proteins.

Authors:Taddeo B, Zhang W, Roizman B,

Journal:Proc Natl Acad Sci U S A

PubMed ID:16477041

The herpes simplex virus 1 ORF UL41 encodes a protein (virion host shutoff or vhs) associated with selective degradation of mRNA early in infection. Some mRNAs, exemplified by GAPDH or beta-actin mRNAs, are degraded rapidly. Others, for example IEX-1 mRNA, are degraded in two stages: whereas the 3' domain disappears ... More

Transcriptional profiling of rhesus monkey embryonic stem cells.

Authors:Byrne JA, Mitalipov SM, Clepper L, Wolf DP,

Journal:Biol Reprod

PubMed ID:16943365

Embryonic stem cells (ESCs) may be able to cure or alleviate the symptoms of various degenerative diseases. However, unresolved issues regarding survival, functionality, and tumor formation mean a prudent approach should be adopted towards advancing ESCs into human clinical trials. The rhesus monkey provides an ideal model organism for developing ... More

Novel GC-rich DNA-binding compound produced by a genetically engineered mutant of the mithramycin producer Streptomyces argillaceus exhibits improved transcriptional repressor activity: implications for cancer therapy.

Authors:Albertini V, Jain A, Vignati S, Napoli S, Rinaldi A, Kwee I, Nur-e-Alam M, Bergant J, Bertoni F, Carbone GM, Rohr J, Catapano CV,

Journal:Nucleic Acids Res

PubMed ID:16571899

The aureolic acid antibiotic mithramycin (MTM) binds selectively to GC-rich DNA sequences and blocks preferentially binding of proteins, like Sp1 transcription factors, to GC-rich elements in gene promoters. Genetic approaches can be applied to alter the MTM biosynthetic pathway in the producing microorganism and obtain new products with improved pharmacological ... More

Endogenous 24(S),25-epoxycholesterol fine-tunes acute control of cellular cholesterol homeostasis.

Authors:Wong J, Quinn CM, Gelissen IC, Brown AJ,

Journal:J Biol Chem

PubMed ID:17981807

Certain oxysterols, when added to cultured cells, are potent regulators of cholesterol homeostasis, decreasing cholesterol synthesis and uptake and increasing cholesterol efflux. However, very little is known about whether or not endogenous oxysterol(s) plays a significant role in cholesterol homeostasis. 24(S),25-Epoxycholesterol (24,25EC) is unique among oxysterols in that it is ... More

Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways.

Authors:Gao X, Wu L, O'Neil RG,

Journal:J Biol Chem

PubMed ID:12738791

The TRPV4 calcium-permeable channel was cloned from mouse kidney M-1 cells, and the effect of temperature modulation on channel gating/activation by physical and chemical signals was evaluated. A TRPV4 cDNA construct with a C-terminal V5 epitope was stably transfected into human embryonic kidney (HEK) 293 and Chinese hamster ovary cells ... More

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