• zebrafish system

MMLV RTase(MMLV反轉錄酶)(熱銷)

ToolsScript M-MLV (MMLV RTase)

MMLV RTase (Moloney Murine Leukemia Virus Reverse Transcriptase)
For first-strand cDNA synthesis and two-step RT-PCR

CAT#TGERA04  20000unit
(功能同 Promega cat#M1701, M-MLV Reverse Transcriptase)

功能:
以ssDNA、ssRNA為模板,合成DNA的反轉錄酶。實驗室中常以MMLV RTase來合成cDNA。

M-MLV介紹:
一般的M-MLV (Moloney Murine Leukemia Virus) 具有以下幾種活性:

  • 依賴於RNA的DNA聚合酶活性
  • 依賴於DNA的DNA聚合酶活性
  • RNase H活性。

由於RNase H能夠降解DNA/RNA中的RNA,因此在第一條鏈cDNA的合成反應中可能會降解RNA/DNA中的模板RNA。

ToolsScrpit M-MLV特性:
此產品以使M-MLV(RNase H)的RNase H失去活性,提高延長的能力,可用於較長的cDNA合成以及全長的cDNA合成等。

 

Description:

ToolsScrpt M-MLV kit Contain

ToolScript M-MLV (200 U/ul) 100 ul
5x First-Strand Buffer 500 ul

 

Storage:
ToolScript MMLV can be stored at –20°C for up to 12 months.

Introduction
ToolScript M-MLV is an RNA-dependent DNA polymerase and consists of a single subunit with a molecular weight of 71kDa. It can be used in cDNA synthesis with RNA or RNA: DNA hybrids as templates. ToolScript M-MLV is the preferred reverse transcriptase for long mRNA templates (>5kb), since its RNase H activity is weaker than commonly used reverse transcriptase. It greatly reduces the degradation of RNA templates and therefore increases the productivity.

Application
Synthesis of first-strand cDNA, One-Step RT-PCR, 3’ and 5’ RACE PCR, prime extension, cDNA library construction, etc.

Unit Definition
One unit is defined as the amount of enzyme that incorporates 1 nmol of dNTPs into acid-insoluble material within 10 min at 37°C with polyA ∙ poly (dT)12-18 as the template-primer.

Protocol
Synthesis of first-strand cDNA 20 μl reaction system can be used for reverse transcription of 1-5 μg total RNA or 50-500 ng mRNA.

  1. Add the following components to a nuclease-free micro-centrifuge tube.
    • 2 μl oligo (dT)12-18 (10 μM), or 2 μl random primers (10 μM), or 2 pmol gene-specific primers
    • 1-5 μg total RNA, or 50-500 ng mRNA;
    • 2 μl dNTP mixture (10 mM total, with neutral PH value);
    • Add RNase-free ddH2O up to 15 μl.
  2. Heat at 70 °C for 5 min, and place the tube immediately on ice for 2 min. Centrifuge briefly and then add 4 μl 5× First-Strand Buffer (with DTT).
    Optional Step: If the amount of starting template is less than 50 ng, 0.5-1 μl RNasin (40 U/μl) should be added.
  3. Add 1 μl ToolScript M-MLV and mix gently by pipetting; when using random primers, incubate the tube at 25°C for 10 min.
  4. Incubate at 42 °C for 50 min.
  5. Heat the sample to 95°C for 5 min to inactivate enzyme. Cool the sample on ice for downstream experiments or store at -20°C immediately.
    If the RNase H is needed, perform the step 6. Or, proceed to step 7 directly.
  6. Add 1 μl RNase H (2 U), incubate at 37°C for 20 min to degrade RNA. Then heat the sample to 95°C for 5 min to inactivate RNase H.
  7. Dilute the reaction system to 50 μl in RNase-free ddH2O. Take 2-5 μl for PCR amplification.

PCR Amplification
Take 10% of the first-strand cDNA synthesis reaction mixture (2 μl) for PCR; increasing amount of cDNA synthesis products not lead to highly efficient DNA amplification and inhibitors presenting in the reverse-transcription products may inhibit the PCR.

  1. Prepare reaction mixture by adding the following components to a microcentrifuge tube.
    Reagents Volume
    10x PCR buffer (200mM Tris-HCL(PH 8.4), 500mM KCl 5ul
    50 mM MgCl2 1.5ul
    Super pure dNTP (2.5mM each) 1ul
    Primer 1 (10 uM) 1ul
    Primer 2 (10 uM) 1ul
    Taq DNA Polymerase (5U/ul) 0.4ul
    cDNA (synthesis reaction mixture) 2ul
    ddH2O up to 50 ul

    Note: To obtain the optimal result, the concentration of MgCl2 should be optimized for individual template-primer combination.

  2. Mix gently and overlay the reaction with one or two drops (~50 μl) of nuclease-free mineral oil to prevent evaporation and condensation. (Mineral oil is not necessary if the thermo cycler has been equipped with hot lid.)
  3. Denature at 94°C for 2 min.
  4. Set 15-40 PCR cycles. The conditions of annealing and denaturation should be optimized for individual primer and template.

 

常見問題

問題: 價格?
回答: 有問有便宜喔!!

 

TOOLS Inc.

Brand: Tools
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  • Light-Plus Biotech Co., Ltd. - 迎光生物技術有限公司。

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