Invitrogen™

GeneRacer™ Kit with SuperScript™ III RT and Zero Blunt™ TOPO™ PCR Cloning Kit for Sequencing

Catalog number:  L150202

Related applications:

PCR & Real-Time PCR

| Cloning

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Description

GeneRacer® is an advanced RACE (rapid amplification of cDNA ends) technique that improves the efficiency of amplifying full-length 5´ and 3´ cDNA ends. With the GeneRacer® Kit you can:

• Generate cDNA from transcripts at least 10 kb in length
• Obtain the full-length 5´ end of rare transcripts at fewer than 30 copies per cell
• Clone the full-length 5´ and 3´ ends to construct complete cDNA sequence

The GeneRacer® Kit is available with SuperScript® III Reverse Transcriptase (RT) for improved amplification of the full-length 5´ end from long and complex mRNA. The RNase H portion of SuperScript® III RT has been mutated to avoid cleaving mRNA during cDNA synthesis. This increases the size and yield of cDNA. SuperScript® III RT is more thermostable than wild-type RTs. This enables reverse transcription at higher temperatures, relaxing secondary structure of complex templates, and allowing cDNA synthesis to go to completion.

How GeneRacer® Works

The GeneRacer® Kit ensures that only transcripts containing full-length cDNA ends are amplified (1,2). Figure 1 outlines how the GeneRacer® Kit works. The advanced protocol starts at the RNA level by specifically targeting only 5´ capped mRNA. In subsequent steps the cap is removed and replaced with the GeneRacer® RNA Oligo. During reverse transcription, the GeneRacer® RNA Oligo sequence is incorporated into the cDNA. Only cDNA that is completely reverse transcribed will contain this known sequence. 5´ RACE PCR is then performed using the GeneRacer® 5´ Primer specific to the GeneRacer® RNA Oligo sequence and a gene-specific primer. The result is amplified DNA that contains the full-length 5´ cDNA sequence.

Sensitivity and Length

To demonstrate the ability of the GeneRacer® Kit to capture the full-length 5fi cDNA end, the 5fi ends of genes with known transcriptional start sites were amplified. Starting with total RNA and following the GeneRacer® protocol, both long transcripts (10 kb) and rare messages present at 0.01%, or 30 copies per cell were amplified (Figure 2).
WARNING: Cancer and Reproductive Harm – www.P65Warnings.ca.gov
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Includes: Each GeneRacer Kit contains the GeneRacer box, an RT box, S.N.A.P. columns, and a TOPO Cloning Kit. The GeneRacer and the RT box contain sufficient reagents for five cDNA reactions plus one control reaction and primers for 50 PCR reactions. Included in the GeneRacer box are the enzymes CIP, TAP, T4 RNA ligase, and their buffers, the GeneRacer RNA Oligo (pre-aliquotted and lyophilized), GeneRacer Primers and Nested Primers, RNaseOUT Recombinant Ribonuclease Inhibitor, phenol/chloroform, mussel glycogen, sterile water, and controls. The SuperScript III RT box includes SuperScript III RT, 5X first-strand buffer, DTT, RNase H, random primers, the GeneRacer Oligo dT Primer, and dNTP mix. The Cloned AMV RT box includes cloned AMV RT, 5X RT buffer, random primers, the GeneRacer Oligo dT Primer, and dNTP mix. A separate bag contains ten S.N.A.P. gel purification columns for gel-purifying PCR products. Store at room temperature. The 10-reaction TOPO Cloning Kit for Sequencing contains two boxes. Competent E. coli .
Quantity: 1 kit
Storage Requirements: The Cloned AMV RT box includes cloned AMV RT, 5X RT buffer, random primers, the GeneRacer Oligo dT Primer, and dNTP mix. Store at -80°C A separate bag contains ten S.N.A.P. gel purification columns, store at room temperature. The 10-reaction TOPO Cloning Kit for Sequencing contains two boxes. Store the TOPO Cloning box at -20°C. Store the competent E. coli box at -70°C. Store all other products in kit at -20°C. All reagents are guaranteed stable for 6 months when properly stored.
Product Line: GeneRacer®, SuperScript™, TOPO®, Zero Blunt®

Contents & storage

Each GeneRacer® Kit contains the GeneRacer® box, an RT box, S.N.A.P.™ columns, and a TOPO® Cloning Kit. The GeneRacer® and the RT box contain sufficient reagents for five cDNA reactions plus one control reaction and primers for 50 PCR reactions. Included in the GeneRacer® box are the enzymes CIP, TAP, T4 RNA ligase, and their buffers, the GeneRacer® RNA Oligo (pre-aliquotted and lyophilized), GeneRacer® Primers and Nested Primers, RNaseOUT™ Recombinant Ribonuclease Inhibitor, phenol/chloroform, mussel glycogen, sterile water, and controls. Store at -20°C. The SuperScript® III RT box includes SuperScript® III RT, 5X first-strand buffer, DTT, RNase H, random primers, the GeneRacer® Oligo dT Primer, and dNTP mix. Store at -20°C. The Cloned AMV RT box includes cloned AMV RT, 5X RT buffer, random primers, the GeneRacer® Oligo dT Primer, and dNTP mix. Store at -80°C. A separate bag contains ten S.N.A.P.™ gel purification columns for gel-purifying PCR products. Store at room temperature. The 10-reaction TOPO® Cloning Kit for Sequencing contains two boxes. Store the TOPO® Cloning box at -20°C. Store the competent E. coli box at -70°C. All reagents are guaranteed stable for 6 months when properly stored.