Thermo Fisher, L150202, GeneRacer™ Kit with SuperScript™ III RT and Zero Blunt™ TOPO™ PCR Cloning Kit for Sequencing

GeneRacer™ Kit with SuperScript™ III RT and Zero Blunt™ TOPO™ PCR Cloning Kit for Sequencing 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 sequenceThe 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™ WorksThe 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 LengthTo 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). Bacterial or Yeast Strain: TOP10
Cloning Method: Zero Blunt TOPO
For Use With (Application): Reverse Transcription
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. CompetentE. coli.
Product Line: GeneRacer™, SuperScript™, TOPO™, Zero Blunt™
Product Type: Cloning Kit
Quantity: 1 Kit
Vector: pCR4Blunt-TOPO
Format: Kit
Unit Size: Each