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高产T7 RNA核磁共振成像(NMR)试剂盒(5F-UTP)
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高产T7 RNA核磁共振成像(NMR)试剂盒(5F-UTP)

高产T7 RNA核磁共振成像(NMR)试剂盒(5F-UTP),HighYield T7 P&L RNA NMR Kit (5F-UTP),设计用于通过 T7 RNA 聚合酶体外转录产生大量 5-荧光修饰的 RNA。更多视频请关注视频号【艾维缔】。哔哩哔哩【IVDSHOW】。抖音【军哥聊表观】。
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Shelf Life: 12 months after date of delivery

Description:

高产T7 RNA核磁共振成像(NMR)试剂盒(5F-UTP)设计用于通过 T7 RNA 聚合酶体外转录产生大量 5-荧光修饰的 RNA。由此产生的内部修饰 (m)RNA 随后可用于通过核磁共振 (NMR) 光谱进行结构测定。

5F-UTP 以一种无干扰的方式(碱基配对特性完好)取代其天然对应物。5F-UTP 修饰同时也是一种敏感的 NMR 报告基团(化学位移弥散比 1H 大)。经修饰的 T7 RNA 聚合酶(T7 P&L RNA 聚合酶)的脯氨酸 266 被亮氨酸(P266L)取代后,转录本的 5'同质性得到了提高,尤其是从 A 启动子 phi2.5 合成的转录本。

该试剂盒含有足够用于 15 个反应(S 袋)或 50 个反应(L 袋)á 20 μl 的试剂(7.5 mM GTP、7.5 mM 5-氟-UTP、7.5 mM ATP、7.5 mM CTP)。使用单核苷酸格式可轻松实现 5-Fluoro-UTP 浓度的个性化优化。
一个 20 μl 的反应在孵育 30 分钟后可产生约 80 μg RNA(1 μg T7 对照模板,1.4 kb RNA 转录本)。不过,产量可能因模板(启动子设计、序列长度、二级结构形成)而异。

Content:
HighYield T7 P&L RNA Polymerase Mix
RNT-202-S: 2x 40 μl incl. RNase inhibitor and 50 % glycerol (v/v)
RNT-202-L: 3x 40 μl incl. RNase inhibitor and 50 % glycerol (v/v)

HighYield T7 Reaction Buffer
1x 200 μl (10x), HEPES-based

ATP - Solution
1x 100 μl (100 mM)

GTP - Solution
1x 100 μl (100 mM)

CTP - Solution
1x 100 μl (100 mM)

UTP - Solution
1x 100 μl (100 mM)

5-Fluoro-UTP
RNT-202-S: 3x 10 μl (100 mM)
RNT-202-L: 8x 10 μl (100 mM)

T7 G-initiating control template (1.4 kbp)
1x 10 μl (200 ng/μl), 1.4 kbp PCR fragment plus T7 class III phi6.5 promotor resulting in ~1400 nt RNA transcript

PCR-grade water
1x 1.2 ml

DTT
1x 100 μl (100 mM)

To be provided by user
T7 Promotor-containing DNA template
RNA purification tools
RNAse-free DNAse I
Important Notes (Read before starting)

Prevention of RNAse contamination
Although a potent RNase Inhibitor is included, creating a RNAse-free work environment and maintaining RNAse-free solutions is critical for performing successful in vitro transcription reactions. We therefore recommend

  • to perform all reactions in sterile, RNAse-free tubes using sterile pipette tips.
  • to wear gloves when handling samples containing RNA.
  • to keep all components tightly sealed both during storage and reaction procedure.

Template requirements

  • Template type: Linearized plasmid DNA or PCR products containing a double-stranded G-initiating T7 class III phi6.5 promotor region upstream of the target sequence.

    Minimum T7 promotor sequences:

    T7 class III phi6.5 promotor (G-initiating)
    5'-TAATACGACTCACTATAGNN…-3’
    Bold: First base incorporated into RNA, NN: ideally CG

  • Template quality: DNA template quality directly influences yield and quality of transcription reaction. Linearized plasmid DNA needs to be fully digested and to be free of contaminating RNase, protein and salts. We recommend selecting restriction enzymes that generate blunt ends or 5'-overhangs and purification by phenol/chloroform extraction. A PCR mixture can be used directly however, better yields will usually be obtained with purified PCR products (e.g. via silica-membrane based purification columns).

In vitro Transcription protocol

The general protocol is set up for 0.5 μg - 1 μg DNA template (refer to section 1.2 regarding template requirements), a final NTP concentration of 7.5 mM and 100 % substitution of UTP by 5-Fluoro-UTP, respectively.

Depending on the RNA sequence and final application, individual reaction optimization may improve product yield and biological function (e.g. variation 5-Fluoro-UTP/UTP ratio , variation of template amount, variation of incubation time).

Component Volume Final conc.
PCR-grade water X μl  
HighYield T7 Reaction Buffer (10x) 2 μl 1x
DTT (100 mM) 2 μl 10 mM
GTP (100 mM) 1.5 μl 7.5 mM
5-Fluoro-UTP (100 mM) 1.5 μl 7.5 mM
CTP (100 mM) 1.5 μl 7.5 mM
ATP (100 mM) 1.5 μl 7.5 mM
Template DNA X μl 1 μg
HighYield T7 P&L RNA Polymerase Mix 2 μl  
Total volume 20 μl  
  • Place HighYield T7 P&L RNA Polymerase Mix on ice.
  • Thaw all remaining components at room temperature (RT), mix by voretexing and spin down briefly.
  • Assemble all components at RT to a nuclease-free microtube (sterile pipette tips) in the following order:
  • Mix PCR-grade water, HighYield T7 Reaction Buffer and DTT by voretexing and spin down briefly.
  • Add nucleotide solutions and template DNA, vortex and spin down briefly.
  • Add HighYield T7 P&L RNA Polymerase Mix vortex and spin down briefly.
  • Incubate for 2h at 37°C in the dark (e.g. PCR cycler). Individual optimization may increase product yield (0.5h–4h at 37°C).

Please note: Reagents for the following steps are not provided within this kit.

DNA template removal
Depending on the down-stream application, removal of template DNA might be required. We recommend a salt-resistant, high efficiency DNAase such as Turbo™DNAse (ThermoFisher). Follow the manufacturer instructions.

RNA purification
Purification of RNA is required for certain applications such as measurement of 5-Fluoro-labeled RNA probe concentration. Spin column purification will remove proteins, salts and unincorporated nucleotides. Please follow the manufacturer instructions and ensure that the columns match with product size and possess a sufficient binding capacity (e.g. RNA Clean & Concentrator™ columns (Zymo Research) or Monarch® RNA Cleanup kit (NEB)). Other RNA purification methods such as LiCl precipitation may work but have not been tested.

Total RNA quantitation
RNA concentration can be determined by absorbance measurement at 260 nm (A260) according to the Law-of-Lambert-Beer (A260 = 1 corresponds to 40 μg/ml ssRNA)

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高产T7 RNA核磁共振成像(NMR)试剂盒(5F-UTP) CNRNT202-01 15次(20ul体系) -20°C
高产T7 RNA核磁共振成像(NMR)试剂盒(5F-UTP) CNRNT202-02 50次(20ul体系) -20°C

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CNRNT202-高产T7 RNA核磁共振成像(NMR)试剂盒(5F-UTP) 操作手册
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保存建议 厂家推荐蓝冰运输。当您收到产品后,按照说明书建议保存于-20°C。

 

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