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.
- 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.
- 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. - Add 1 μl ToolScript M-MLV and mix gently by pipetting; when using random primers, incubate the tube at 25°C for 10 min.
- Incubate at 42 °C for 50 min.
- 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. - 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.
- 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.
- 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. - 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.)
- Denature at 94°C for 2 min.
- 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!!
Brand: Tools
Distributor:
- GENDANIO Ocean Technology Co., Ltd. - 鈞達海洋科技有限公司。
- Light-Plus Biotech Co., Ltd. - 迎光生物技術有限公司。
More detail information: