synthesis with dUTP from First Strand cDNA: ..
The synthesis of DNA from an RNA template, via reverse transcription, produces complementary DNA (cDNA). Reverse transcriptases (RTs) use an RNA template and a short primer complementary to the 3' end of the RNA to direct the synthesis of the first strand cDNA, which can be used directly as a template for the Polymerase Chain Reaction (PCR). This combination of reverse transcription and PCR (RT-PCR) allows the detection of low abundance RNAs in a sample, and production of the corresponding cDNA, thereby facilitating the cloning of low copy genes. Alternatively, the first-strand cDNA can be made double-stranded using DNA Polymerase I and DNA Ligase. These reaction products can be used for direct cloning without amplification. In this case, RNase H activity, from either the RT or supplied exogenously, is required.
strand for double stranded C-DNA without using kit.
Many RTs are available from commercial suppliers. and Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase are RTs that are commonly used in molecular biology workflows. lacks 3´ → 5´ exonuclease activity. is a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability. It can be used to synthesize first strand cDNA at higher temperatures than the wild-type M-MuLV. The enzyme is active up to 50°C, providing higher specificity, higher yield of cDNA and more full-length cDNA product, up to 12 kb in length.
Mint-2 cDNA synthesis kit is designed to synthesize full-length-enriched double stranded (ds) cDNA from total or poly(A)+ RNA. Synthesized cDNA can be used for construction of cDNA libraries, subtractive hybridization (SSH), high-throughput sequencing on the next generation sequencing platforms (Roche/454, ABI/SOLiD or Illumina/Solexa), and other applications.