Sigma-Aldrich Transcriptor Reverse Trans

Recombinant

expressed inE. coli

Quality Level

100

Assay

≥90% (SDS-PAGE)

Form

liquid

Usage

sufficient for 25 reactions
sufficient for 50 reactions
sufficient for 500 reactions

Specific Activity

0.05 U/mg

Feature

dNTPs included: no
hotstart: no

Manufacturer/Tradename

Roche

Packaging

pkg of 200 reactions (03531287001)
pkg of 25 reactions (03531317001)
pkg of 50 reactions (03531295001)

Technique(S)

RT-PCR: suitable
RT-qPCR: suitable

Related Categories

PCR Reagents & Kits

Roche® Life Science Products

General Description

A recombinant reverse transcriptase for the robust transcription of RNA fragments up to 14 kb used in two-step RT-PCR on thermal cyclers and real-time PCR instruments.In retroviruses like the human immunodeficiency virus type 1 (HIV-1), reverse transcriptase (RT) is the core enzyme. HIV-1 RT is made of two subunits of 66 kDa and 51 kDa (p66 and p5l).A human endogenous retrovirus of the HERV-K family codes for reverse transcriptase (RT) enzyme.

Application

Transcriptor Reverse Transcriptase is designed to transcribe RNA (mRNA, total RNA, viral RNA, andin vitro-transcribed RNA) from a variety of sources, using conventional thermal cyclers and real-time PCR instruments (e.g.,the LightCycler^®Instruments) for the following applications:

• Synthesis of first-strand cDNA for use in subsequent amplification reactions
• RT-PCR of GC-rich RNA templates
• Cy3, Cy5, DIG, biotin, and aminoallyl labeling during cDNA synthesis
• Retrieving and cloning the 5′ and 3′ termini of mRNA by RACE
• Generation of cDNA libraries with large inserts
• Dideoxy DNA sequencing
• RNA sequencing
• 3′-end labeling of DNA fragments
• Generation of single-stranded probes for genomic footprints
• In the reverse transcription of RNA from human Papillomavirus E6,cortical and striatal tissues, muscle biopsies of Becker muscular dystrophy samples[5] and miRNA stem-loop from oocytes[6] and human cerebral microvascular endothelial cells (hCMVECs)[7]

Biochem/Physiol Actions

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is essential for the catalytic conversion of single-stranded viral RNA into the double-stranded linear DNA that is integrated into host cell chromosomes.

Features And Benefits

• Achieve high sensitivity in two-step RT-PCR.

Transcriptor Reverse Transcriptase is used in conventional thermal cyclers and real-time PCR instruments (e.g., the LightCycler^®Instruments).

• Obtain more full-length transcripts - up to 14 kb.

cDNA libraries with large inserts can be generated.

• Reverse transcribe difficult templates.

The enzyme works well at elevated temperatures, thereby overcoming RNA secondary structure (e.g., GC-rich RNA templates) and facilitating optimal reaction conditions.

• Efficiently label cDNA.

Cy3-, Cy5-, DIG-, biotin-, or aminoallyl-labeled nucleotides are incorporated during cDNA synthesis.

Components

• Transcriptor Reverse Transcriptase, in storage buffer
• Transcriptor RT buffer, 5x concentrated

Quality

Each lot of Transcriptor Reverse Transcriptase is routinely function tested in RT-PCR:

• with a conventional thermal cycler to detect a 10-kb fragment from the human dystrophin gene, starting from human skeletal muscle total RNA
• with the LightCycler^®Instrument to detect 5 x 10²to 5 x 10⁶copies ofin vitro-transcribed human PBGD RNA. The results are defined in fixed crossing points and fixed fluorescence intensity.

Preparation Note

Volume activity:20 U/μl.
Transcription of long, rare, or difficult RNA targets
Transcriptor Reverse Transcriptase is recommended for RT-PCR of:

• long targets, because it can transcribe up to 14 kb RNA templates
• rare targets, because it has high sensitivity
• GC-rich targets, because it can operate at high temperatures (up to +65^oC) to eliminate problems associated with extensive secondary structure

Labeling for many applications
Transcriptor Reverse Transcriptase is also recommended for preparing labeled cDNA, since it accepts a wide variety of modified nucleotides (including Cy3-, Cy5-, DIG-, Biotin-, or aminoallyl-labeled dNTPs).
Reaction requirements
Transcriptor accepts ssRNA and ssDNA templates and requires a primer for transcription.

Storage And Stability

Avoid repeated freezing and thawing.

Other Notes

For life science research only. Not for use in diagnostic procedures.LightCycler is a registered trademark of Roche