SuperMix de síntesis de primera cadena SuperScript™ III

Citas y referencias (4)

Invitrogen™

SuperMix de síntesis de primera cadena SuperScript™ III

Fórmula SuperMix optimizada para síntesis de la primera cadena del ADNc a partir de poli(A)+ purificado O ARN total

Número de catálogo	Cantidad
18080400	50 rxns

Número de catálogo 18080400

Precio (MXN)

Cantidad:

50 rxns

Pedido a granel o personalizado

The SuperScript III First-Strand Synthesis SuperMix is an optimized formulation for first-strand cDNA synthesis from purified poly(A)⁺ or total RNA. RNA targets from 100 bp to >12 kb can be detected with this system. The amount of starting material can vary from 0.1 pg to 5 μg of total RNA.

This kit includes SuperScript III/RNaseOUT enzyme mix, 2X First-Strand reaction mix, and an annealing buffer. SuperScript III Reverse Transcriptase is a version of MMLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability. The enzyme can be used to synthesize cDNA at a temperature range of 45–55°C, providing increased specificity, higher yields of cDNA, and more full-length product than many previous reverse transcriptases.

El sistema de síntesis para primera cadena SuperScript™ III SuperMix es una formulación optimizada del SuperMix para la síntesis de la primera cadena de cADN a partir de poli (A)⁺ purificado del ARN total. Con este sistema, pueden detectarse objetivos de ARN de 100 bp a >12 kb. La cantidad de material inicial puede variar de 0,1 pg a 5 µg de ARN total.

Uso del sistema de síntesis para primera cadena SuperScript™ III SuperMix
El kit incluye la mezcla de enzimas SuperScript™ III o RNaseOUT™, la mezcla para la reacción de síntesis de primera cadena 2X y un tampón de recocido. La transcriptasa inversa SuperScript™ III, incluida en la mezcla de enzimas, es una versión de la transcriptasa inversa de virus de leucemia murina Moloney (M-MLV) que se ha diseñado para reducir la actividad de la ribonucleasa (ARNasa) H y proporcionar mayor estabilidad térmica. La enzima se puede utilizar para sintetizar ADNc en un intervalo de temperatura de 45–55 °C, y proporcionar una mayor especificidad, mayor producción de ADNc y más cantidad de producto de cadena completa que otras transcriptasas inversas. Como la transcriptasa inversa SuperScript™ III no se ve significativamente inhibida por el ARN de transferencia y ribosómico, se puede emplear para sintetizar ADNc de ARN total. El inhibidor de ARNasa recombinante RNaseOUT™ se incluye en la mezcla de enzimas para la protección contra la degradación del ARN objetivo por la contaminación de la ribonucleasa. La mezcla de reacción de primera cadena 2X incluye 10 mm de MgCl₂ y 1 mm de cada desoxinucleótido (dNTP) en una solución de tampón que se ha optimizado para la síntesis de primera cadena de ADNc. El tampón de hibridación se utiliza en el paso inicial de anillaje del cebador. También se suministran tubos independientes de oligo(dT)₂₀ y hexámeros al azar. La síntesis de ADNc se puede realizar con ARN total o ARN seleccionado mediante poli (A)⁺ con cebadores aleatorios oligo(dT) o un cebador específico del gen.

Para uso exclusivo en investigación. No apto para uso en procedimientos diagnósticos.

Especificaciones

Tipo de producto finalPrimera cadena de ADNc

FormatoMezcla maestra

N.º de reacciones50 reacciones

Temperatura óptima de reacción50 °C

CebadorCebadores aleatorios, cebadores oligo dT

Cantidad50 rxns

Formato de reacciónMezcla maestra

Tipo de reactivoTranscripción reversa

Transcriptasa inversaSuperScript III

Condiciones de envíoHielo seco

Material de partidaARN

TécnicaTranscripción reversa

Para utilizar con (aplicación)PCR en tiempo real (PCRq)

GC-Rich PCR PerformanceAlto

Velocidad de reacción50 min

Unit SizeEach

Contenido y almacenamiento

• Mezcla de reacción 2X, 500 μl
• Mezcla de enzimas SuperScript™ III, 100 μl
• Oligo(dT)₂₀, 50 μl total (50 μM)
• Hehámeros aleatorios, 50 μl (50 ng/μl)
• Tampón de recocido, 50 μl

Almacenar a –20 °C.

Preguntas frecuentes

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.

I'm setting up my RT reaction and am trying to decide whether I should use random primers, oligo(dT) primer, gene-specific primer, or oligo(dT)/random mix primers. What would you suggest?

Random primers are the best choice for degraded RNA, RNA with heavy secondary structure, non-polyadenylated RNA, or prokaryotic RNA. It is recommended only for two-step RT-PCR, and typically gives the highest yields, although the cDNA may not necessarily be full length. Oligo(dT) primers are good to use when trying to recover full-length cDNA from 2-step RT-PCR. The reaction is influenced by secondary structure and RNA quality. Gene specific primers should be used for very specific, mainly one-step RT-PCR reactions.

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

View all SuperMix de síntesis de primera cadena SuperScript™ III FAQs

Citations & References (4)

Citations & References

Abstract

TDAG51 is an ERK signaling target that opposes ERK-mediated HME16C mammary epithelial cell transformation.

Authors:Oberst MD, Beberman SJ, Zhao L, Yin JJ, Ward Y, Kelly K,

Journal:BMC Cancer

PubMed ID:18597688

'INTRODUCTION: Signaling downstream of Ras is mediated by three major pathways, Raf/ERK, phosphatidylinositol 3 kinase (PI3K), and Ral guanine nucleotide exchange factor (RalGEF). Ras signal transduction pathways play an important role in breast cancer progression, as evidenced by the frequent over-expression of the Ras-activating epidermal growth factor receptors EGFR and ... More

Elevated serum IL-10 levels in diffuse large B-cell lymphoma: a mechanism of aberrant JAK2 activation.

Authors:Gupta M, Han JJ, Stenson M, Maurer M, Wellik L, Hu G, Ziesmer S, Dogan A, Witzig TE,

Journal:Blood

PubMed ID:22323454

'Cytokines are deregulated in cancers and can contribute to tumor growth. In patients with diffuse large-cell lymphoma (DLBCL), we observed higher levels of JAK/STAT pathway-related serum cytokines (ie, IL-6, IL-10, epidermal growth factor, and IL-2) compared with controls. Of these, only IL-10 activated the JAK2 pathway in lymphoma cells in ... More

Interactive effects of neurohypophyseal neuropeptides with receptor antagonists on passive avoidance behavior: mediation by a cerebral neurohypophyseal hormone receptor?

Authors:de Wied D, Elands J, Kovács G,

Journal:Proc Natl Acad Sci U S A

PubMed ID:1847526

The neurohypophyseal neuropeptides (Arg8)-vasopressin (AVP) and [pGlu4,Cyt6]AVP-(4-8) (where pGlu is pyroglutamic acid and Cyt is cystine) facilitate the retention of one-trial-learning passive avoidance behavior in rats when administered into the cerebral ventricle immediately after the learning trial. The fragment [pGlu4,Cyt6]AVP-(4-8) was considerably more effective than AVP. Oxytocin (OXT) and [pGlu4,Cyt6]OXT-(4-8) ... More

Flavivirus NS4A-induced autophagy protects cells against death and enhances virus replication.

Authors:McLean JE, Wudzinska A, Datan E, Quaglino D, Zakeri Z,

Journal:J Biol Chem

PubMed ID:21511946

Flaviviruses include the most prevalent and medically challenging viruses. Persistent infection with flaviviruses of epithelial cells and hepatocytes that do not undergo cell death is common. Here, we report that, in epithelial cells, up-regulation of autophagy following flavivirus infection markedly enhances virus replication and that one flavivirus gene, NS4A, uniquely ... More