上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。
产品信息
FCE retains significant capping activity at low temperatures and tolerates reaction temperatures up to 55°C. In many cases, 1 µl of FCE (25 units) can cap over 100 µg of RNA in 1 hour at 37°C. GTP and S-adenosylmethionine (SAM) are required for capping activity and are included with the enzyme.
Source: An E. coli strain that carries a plasmid encoding the Faustovirus Capping Enzyme with a C-terminal His-tag.
B. mRNA capping by FCE at 37°C and 42°C. 200 μg (~350 picomoles, 7 μM) of a 1.77 kb FLuc transcript having 5′-UTR sequences as indicated were treated with a limiting amount (25 units, 1 picomole or 20 nM) of FCE for 1 hour at 37°C or 42°C. Note that this is less than our recommended amount of enzyme highlighting the potential benefits of workflow optimization. All capping reactions were performed in 50 μl reactions containing 0.1 mM SAM, and 0.5 mM GTP, 1X FCE Capping Buffer for FCE reactions or 1X Capping Buffer for VCE reactions. Following capping reactions, mRNA capping was measured using targeted RNase H cleavage and LC-MS.
- 产品类别:
- RNA Capping
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产品组分信息
本产品提供以下试剂或组分：
NEB # 名称 组分货号 储存温度 数量 浓度 -
M2081S -20 Faustovirus Capping Enzyme M2081SVIAL -20 1 x 0.02 ml 25,000 units/ml FCE Capping Buffer B2181AVIAL -20 1 x 0.5 ml 10 X S-adenosylmethionine (SAM) B9003SVIAL -20 1 x 0.1 ml 32 mM GTP N2080AVIAL -20 1 x 0.05 ml 10 mM
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M2081L -20 Faustovirus Capping Enzyme M2081LVIAL -20 1 x 0.1 ml 25,000 units/ml FCE Capping Buffer B2181AVIAL -20 1 x 0.5 ml 10 X S-adenosylmethionine (SAM) B9003SVIAL -20 1 x 0.1 ml 32 mM GTP N2080AAVIAL -20 1 x 0.25 ml 10 mM
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特性和用法
单位定义
One unit of Faustovirus Capping Enzyme is defined as the amount of enzyme required to convert 75 pmol of a 20-mer transcript to Cap-0 RNA in 30 minutes at 37°C.
反应条件
1X FCE Capping Buffer
Supplement with 0.5 mM GTP 和 0.1 mM S-adenosylmethionine (SAM)
Incubate at 37°C1X FCE Capping Buffer
50 mM Tris-HCl
5 mM KCl
1 mM MgCl2
1 mM DTT
0.02% Poloxamer 188
(pH 8 @ 25.0°C)贮存溶液
40 mM Tris-HCl
100 mM NaCl
50 mM Arginine
0.1 mM TCEP
50% Glycerol
pH 8 @ 25°C热失活
70°C for 10 minutes
Addition of EDTA to 5 mM is recommended to avoid RNA hydrolysis.
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优势和特性
Features
- Experience improved capping efficiency, even on difficult substrates
- Achieve robust capping with less enzyme
- Set up reactions under a broad temperature range, for added flexibility
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相关产品
相关产品
- T2030 Monarch RNA Cleanup Kit 10 ug
- T2040 Monarch RNA Cleanup Kit 50 ug
- T2050 Monarch RNA Cleanup Kit 500 ug
- e2040-hiscribe-t7-high-yield-rna-synthesis-kit
- e2050-hiscribe-t7-quick-high-yield-rna-synthesis-kit
- mRNA 帽结构 2-O-甲基转移酶
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参考文献
- Ramanathan, A. et al. (2016). Nucleic Acids Res . 44 (16), 7511–7526.
- Furuichi, Y., LaFiandra, A. & Shatkin, A. (1977). Nature. 266, 235-239.
- Schlee, M., Hartmann, G. (2016). Nat Rev Immunol . 16, 568-580.
操作说明、说明书 & 用法
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操作说明
- Faustovirus Capping Enzyme FCE Standard Capping Protocol (NEB #M2081)
- One-pot synthesis of Cap-1 Synthesis with Faustovirus Capping Enzyme (FCE) and Cap 2´-O-methyltransferase (NEB #M2081, #M0366)
- Cap-0 synthesis using Faustovirus Capping Enzyme (FCE) (NEB #M2081)
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应用实例
- Scaling of High-Yield In vitro Transcription Reactions for Linear Increase of RNA Production
FAQs & 问题解决指南
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FAQs
- What are Cap-0 and Cap-1?
- Can Faustovirus Capping Enzyme (NEB #M2081) use cap analogs in a capping reaction?
- Can Faustovirus Capping Enzyme (NEB #M2081) use GTP analogs in a capping reaction?
- Is Faustovirus Capping Enzyme sensitive to secondary structure?
- Is Faustovirus Capping Enzyme active at temperatures other than 37°C?