GeneRacer® Kit with SuperScript® III RT and TOPO TA Cloning® Kit for Sequencing

Catalog number: L150201

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

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Description

The GeneRacer™ Kit provides a method to obtain full-length 5' and 3' ends of cDNA using known cDNA sequence from expressed sequence tags (ESTs), subtracted cDNA, differential display, or library screening. The kit ensures the amplification of only full-length transcripts via elimination of truncated messages from the amplification process. RACE PCR products can be quickly and easily cloned using either the Zero Blunt® TOPO® PCR Cloning Kit for Sequencing (blunt-end PCR products) or the TOPO TA Cloning® for Sequencing Kit (PCR products with 3' A-overhangs). Using the protocols provided, the cDNA ends of rare (30 copies/cell) and long (9 kb) transcripts can be amplified and sequenced starting from 1 µg of total RNA. 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 sequenceSuperScript® III RT
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 the GeneRacer® Kit works
The GeneRacer® Kit ensures that only transcripts containing full-length cDNA ends are amplified (see figure). 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 5´ cDNA end, the 5´ 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 (see figure).
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Product Size: 1 kit

Contents & storage

Store each module as indicated:

GeneRacer™ Module (-20°C):

• 2 × 1.5 ml Sterile Water

• 24 µl RNaseOut™

• 6 µl each Calf Intestinal Phosphatase (CIP), CIP Buffer, Tobacco Acid Pyrophosphatase (TAP), 10X TAP Buffer, T4 RNA Ligase, 10X T4 RNA Ligase Buffer, and 10 mM ATP

• 6 × 250 ng GeneRacer™ RNA Oligo

• 2 × 1 ml Phenol/Chloroform

• 36 µl Mussel Glycogen

• 200 µl Sodium Acetate (3 M)

• 225 µl each GeneRacer™ 5′ Primer, 5′ Nested Primer, 3′ Primer, and 3′ Nested Primer

• 20 µl Control HeLa Total RNA (500 ng/µl)

• 15 µl each Control Primer A and Control Primer B.1

SuperScript™ III RT Module (-20°C)

• 6 µl SuperScript™ III Reverse Transcriptase (200 U/µl)

• 24 µl 5X First Strand Buffer

• 15 µl DTT (0.1 M)

• 6 µl RNaseH (2 U/µl)

• 6 µl Random Primers (100 ng/µl)

• 6 µl GeneRacer™ Oligo dT Primer (900 ng/µl)

• 6 µl dNTP Mix (10 mM each)

10 S.N.A.P.™ Columns (room temperature)

TOPO TA Cloning® Kit for Sequencing (-20°C)

• Sufficient reagents and One Shot® TOP10 Chemically Competent E. coli (store at -80°C) to clone 10 GeneRacer™ PCR products

Documents

Manuals & protocols

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