GeneRacer® Kit with AMV RT and TOPO TA Cloning® Kit for Sequencing

Catalog number: L150001

Invitrogen™  Related applications: Reverse Transcription | cDNA Libraries & Library Construction

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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 up to 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).
For Research Use Only. Not for use in diagnostic procedures.


Product Size: 1 kit

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.


Manuals & protocols

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