ProtoScript®First Strand cDNA Synthesis Kitfeatures two optimized mixes, M-MuLV Enzyme Mix and M-MuLV Reaction Mix. M-MuLVEnzyme Mix combines M-MuLV Reverse Transcriptase and Murine RNase Inhibitor,while M-MuLV Reaction Mix contains dNTPs and an optimized buffer. The kit alsocontains two optimized primers for reverse transcription and nuclease-freewater. An anchored oligo-dT primer [d(T)23VN] forces the primer toanneal to the beginning of the polyA tail. The optimized Random Primer Mixprovides random and consistent priming sites covering the entire RNA templateincluding both mRNAs and non-polyadenylated RNAs. The first strand cDNA productgenerated is more than 10 kb (Figure 1).General Information forSuccessful cDNA Synthesis:Template RNAIntact RNA of high purity is essential for sensitive RT-PCR detection. RNAshould have a minimum A260/A280 ratio of 1.7 orhigher.Either total RNA or mRNA can be used in the reversetranscription reaction. Total RNA is generally sufficient for most RT-PCRanalyses. However, if desired mRNA can be easily obtained using a PolyA SpinmRNA Isolation Kit (NEB #S1560) or Magnetic mRNAIsolation Kit (NEB #S1550).The amount ofRNA required for detection depends on the abundance of the transcript ofinterest. In general 1 ng to 1 μg total RNA or 0.1-100 ng mRNA arerecommended.First Strand cDNA SynthesisReactionDenaturation of RNA and primer at 70°C for 5 minutes canremove secondary structures that may impede long cDNA synthesis. However, thisstep can be omitted in some cases (unpublished results).We recommendincubation at 42°C for one hour for maximum yield and length. However, manytargets can be detected after a much shorter incubation time. For example, 5minutes incubation is enough for a 2 kb cDNA synthesis.Choice ofPrimers for Reverse TranscriptionOligo d(T) priming is preferredfor most applications because it ensures that all cDNA copies terminate at the3´ end of the mRNA and produces the longest contiguous cDNA. An anchoredoligo-d(T) primer [d(T)23VN] forces the primer to anneal to the startof the polyA tail, thereby preventing priming at internal sites in the polyAtail (1). However, two other priming choices are possible if desired.The Random Primer Mix is an optimized mix of hexamer andd(T)23VN primers. It provides random priming sites covering theentire RNA templates including both mRNAs and non-polyadenylated RNAs (such asribosomal RNAs). The Random Primer Mix yields shorter cDNAs on average and canbe used for the detection of multiple short RT-PCR products. Random Primer Mixoffers good performance in a wide range of RNA templates.When agene-specific primer is used in a cDNA synthesis reaction, the cDNA product canbe used only for amplification of that transcript. This priming method givesgood results when the amount of RNA is limiting (below 10 ng) and only oneparticular cDNA is desired.Recommended primer concentration:PRIMER-- Final conc.OLIGO d(T)23VN -- -- 5 μM RANDOM PRIMER MIX --6 μM SPECIFIC PRIMER-- 0.1–1 μMFigure 1:First strand cDNA synthesis was carried out with 1X M-MuLV Enzyme Mix at 42°C using 2 μg of human spleen total RNA. Negative control reactions were carried out with 1X M-MuLV Reaction Mix. A fraction of the first strand cDNA product was used to amplify sequences specific for three different messenger RNAs using 1X LongAmp™ Taq 2X Master Mix (NEB #M0287 ). Lane 1: 1.1 kb of beta-actin gene. Lane 2: noRT control of 1.1 kb of beta-actin gene. Lane 3: 4.7 kb of Xrn-1 gene. Lane 4: noRT control of 4.7 kb of Xrn-1 gene. Lane 5: 9.8 kb of guanine nucleotide exchange factor p532. Lane 6: noRT control of 9.8 kb of guanine nucleotide exchange factor p532. Marker M is 2-Log DNA Ladder (NEB #N3200 ).
This product is related to the following categories:
RT-PCR Products,
Reverse Transcriptases & RT-PCR Products,
cDNA Synthesis & Reverse Transcriptases Products
This product can be used in the following applications:
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