Invitrogen™

FirstChoice™ RLM-RACE Kit with Manual

Catalog number:  AM1700M

Related applications:

cDNA Libraries & Library Construction

| Reverse Transcription

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Description

The FirstChoice® RLM-RACE Kit is designed to amplify cDNA only from full-length, capped mRNA, usually producing a single band after PCR. This kit is a major improvement over the basic rapid amplification of cDNA ends (RACE) protocol. The RLM-RACE procedure selects only full-length mRNA—no rRNA, tRNA or degraded RNA—and facilitates the cloning of sequences from the 5' ends of messages.

Features of the FirstChoice® RLM-RACE Kit:

• From RNA to PCR product in less than a day
• Yields single, specific product from rare transcripts
• Selects 5' and/or 3' ends of true messages
• Efficient—all enzymatic reactions are optimized to ensure detection of even the rarest mRNA

RACE
Rapid amplification of cDNA ends (5'-RACE) is a polymerase chain reaction-based technique developed to facilitate the cloning of sequence from the 5'-ends of messages. The FirstChoice® RLM-RACE Kit is a major improvement to the basic RACE protocol.

Selects full-length mRNA—no rRNA, tRNA or degraded mRNA
In the FirstChoice® RLM-RACE procedure, full-length mRNAs are selected by treating total or poly(A) RNA with calf intestinal phosphatase (CIP) to remove the 5'-phosphate from all molecules which contain free 5'-phosphates (ribosomal RNA, fragmented mRNA, tRNA, and contaminating genomic DNA). Full-length mRNAs are unaffected. The RNA is then treated with tobacco acid pyrophosphatase (TAP) to remove the cap structure from the full-length mRNA leaving a 5'-monophosphate. A synthetic RNA adapter is ligated to the RNA population—only molecules containing a 5'-phosphate, the uncapped, full-length mRNAs, will accept the adapter. Random-primed, reverse transcription reactions and nested PCR are then performed to amplify the 5'-end of your specific transcript.

From RNA to PCR in less than a day
Each step of the FirstChoice® RLM-RACE procedure has been optimized, so you can complete all the enzymatic reactions and even start PCR in no more than 5 hours. The FirstChoice® RLM-RACE Kit contains reagents and enzymes to produce 5 RLM-RACE-ready cDNA preparations, in addition to primers and nested RACE adapter primers for 100 PCR reactions. Reverse transcription reagents are also included. Each kit contains control RNA and primers to test the kit's performance along with a detailed Instruction Manual. SuperTaq™ Thermostable Taq DNA Polymerase is available separately. For optimal amplification of fragments ≥1 kb, use SuperTaq-Plus. Also included are a 3' RACE Adapter and Primers for 3' RACE.
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Final Product Type: cDNA
For Use With (Application): cDNA Libraries & Library Construction
Format: Kit
Includes: Exo– Klenow, 10X Decamer Solution, 5X Reaction Buffer minus dATP, 5X Reaction Buffer minus dCTP, DECAtemplate GAPDH-M, and 0.5 M EDTA should be stored at -20°C. Nuclease-free water may be stored at any temperature and manual
Product Type: RLM-RACE Kit
Quantity: 1 Kit
Optimal Reaction Temperature: 42° C
Reaction Format: Separate Components
Reverse Transcriptase: M-MLV
Product Line: Ambion™, FirstChoice™

Contents & storage

Exo– Klenow, 10X Decamer Solution, 5X Reaction Buffer minus dATP, 5X Reaction Buffer minus dCTP, DECAtemplate™ GAPDH-M, and 0.5 M EDTA should be stored at –20°C. Nuclease-free water may be stored at any temperature.