Invitrogen™ GeneRacer™ Kit with AMV RT and TOPO TA Cloning™ Kit for Sequencing

Advanced RACE (rapid amplification of cDNA ends) technique that improves efficiency of amplifying full-length 5' and 3' cDNA ends

Supplier:  Invitrogen™ L150001

Catalog No. L150001

Description

The GeneRacer Kit is available with SuperScriptIII Reverse Transcriptase (RT) for improved amplification of the full-length 5' end from long and complex mRNA. The RNase H portion of SuperScriptIII RT has been mutated to avoid cleaving mRNA during cDNA synthesis. This increases the size and yield of cDNA. SuperScriptIII 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.

• Generate cDNA from transcripts at least 10kb 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

• Ensures that only transcripts containing full-length cDNA ends are amplified (1,2)
• Advanced protocol starts at RNA level by specifically targeting only 5' capped mRNA
• In subsequent steps, cap is removed and replaced with GeneRacer RNA Oligo
• During reverse transcription, GeneRacer RNA Oligo sequence is incorporated into cDNA
• Only cDNA that is completely reverse transcribed will contain this known sequence
• 5' RACE PCR is then performed using GeneRacer 5' Primer specific to GeneRacer RNA Oligo sequence and gene-specific primer
• Result is amplified DNA that contains the full-length 5' cDNA sequence
• To demonstrate the ability of GeneRacer Kit to capture full-length 5fi cDNA end, 5fi ends of genes with known transcriptional start sites were amplified
• Starting with total RNA and following GeneRacer protocol, both long transcripts (10 kb) and rare messages present at 0.01%, or 30 copies per cell were amplified

Cloning, PCR and Real-Time PCR, Reverse Transcription, cDNA Libraries and Library Construction

Specifications

• Bacterial or Yeast Strain: TOP10
• Content And 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.
• For Use With (Application): Reverse Transcription
• Product Line: GeneRacer™, TA Cloning™, TOPO™
• Quantity: 1 Kit

• Cloning Method: TOPO TA
• Format: Kit
• 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. 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.
• Product Type: Cloning Kit
• Vector: pCR4-TOPO TA

For Research Use Only. Not for use in diagnostic procedures.