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Shelf Life: 12 months after date of delivery
Description:
高产 T7 mRNA 合成试剂盒（m5CTP）设计用于通过 T7 RNA 聚合酶体外转录产生大量 5-甲基胞嘧啶修饰的（m）RNA。由此产生的 5'-capped 和内部修饰的 (m)RNA 随后可用于显微注射、转染或体外翻译实验。
研究表明，5-甲基胞嘧啶修饰可增加（m）RNA 的稳定性并降低免疫原性。5'-cap 结构可进一步提高翻译效率。5'-capping 可通过以下方式实现：a) 使用cap analoga（如 ARCA）进行共转录加帽；或 b) 使用加帽酶进行转录后加帽。
试剂盒中的试剂足够用于 15 个反应（S 袋）或 50 个反应（L 袋）à 20 μl （7.5 mM GTP、7.5 mM UTP、7.5 mM ATP、7.5 mM 5-甲基-CTP）。使用单核苷酸格式可轻松实现 5-Methyl-CTP 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 100-130 μg RNA（1 μg T7 对照模板，1.4 kb RNA 转录本）。不过，产量可能会因模板（启动子设计、序列长度、二级结构形成）的不同而有所变化）。
Content:
HighYield T7 RNA Polymerase Mix
RNT-110-S: 2x 40 μl incl. RNase inhibitor and 50 % glycerol (v/v)
RNT-110-L: 3x 40 μl incl. RNase inhibitor and 50 % glycerol (v/v)HighYield T7 Reaction Buffer
1x 200 μl (10x), HEPES-basedATP - 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-Methyl-CTP
RNT-110-S: 3x 10 μl (100 mM)
RNT-110-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
T7 A-initiating control template (1.4 kbp)
1x 10 μl (200 ng/μl), 1.4 kbp PCR fragment plus T7 class II phi2.5 promotor (A-initiating) resulting in ~1400 nt RNA transcriptPCR-grade water
1x 1.2 mlDTT
1x 100 μl (100 mM)To be provided by user
T7 Promotor-containing DNA template
RNA purification tools
RNAse-free DNAse IImportant 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/chloro¬form 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).
- mRNA production: For the production of functional mRNA, the DNA template needs to encode the following structural features e.g. 3’-UTR, 5’-UTR, correctly orientated target sequence and poly(A)-tail. Alternatively, poly (A)-tailing can post-transcriptionally be performed with Poly(A) polymerase.
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 CTP by 5-Methyl-CTP, respectively.
Depending on the RNA sequence and final application, individual reaction optimization may improve product yield and biological function (e.g. variation 5-Methyl-CTP/CTP 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 UTP (100 mM) 1.5 μl 7.5 mM 5-Methyl-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 RNA Polymerase Mix 2 μl Total volume 20 μl - Place HighYield T7 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 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.Removal of 5'-triphosphate groups
5'-ends of in vitro phosphorylated RNAs carry a triphosphate group that is known to trigger RIG-1 mediated innate immune response in mammalian cells[1,2]. Removal with phosphatases (e.g. CIP) before final purification is therefore recommended for RNA probes intended for transfection experiments. Please refer to the following references for more detailed information: [1],[2].(m)RNA purification
Purification of (m)RNA is required prior to transfection or (m)RNA quantitation by absorbance measurement. 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.(m)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). -
产品组分
内容 型号
规格 储存温度
高产T7 mRNA合成试剂盒 (m5CTP)
CNRNT110-01 15次x20ul -20°C 高产T7 mRNA合成试剂盒 (m5CTP)
CNRNT110-02 50次x20ul -20°C 操作手册
1 1 常温
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注意事项
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。 -
FAQ
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[1] Wienert et al. (2018) In vitro transcribed guide RNAs trigger an innate immune response via RIG-I pathway. PLoS Biol. 16 (7) :e2005840.
[2] Kim et al. (2018) CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 28 (3):367.
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