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This kit is using T7 RNA Polymerase to contain a linearized plasmid DNA, a PCR product or synthetic DNA containing a T7 promoter, and the synthesis of RNA is transcribed in vitro. This kit optimizes the RNA in vitro transcriptional reaction system, which can simply quickly obtain a large number of RNA molecules. If the modified nucleotide is added in the substrate, the reactive nucleotide can be prepared, and RNA labeled can be prepared. This kit is mainly used in in vitro translation, RNASE protection experiment, hybrid probe tag, RNA shear and other biological experiments. A template input of 1 μg in the reaction system can produce an RNA greater than 100 μg, suitable for the preparation of RNA of length 100-5000 NT.
Storage and transportation
Transport with wet ice; storage at -20°C, valid for 12 months.
1. Template preparation:
a. Use the plasmid of the T7 promoter as the template: In order to obtain RNA of a specific length, the plasmid template must be completely linearized (it needs to be purified as a template), and the linearized plasmid should ensure that the double strands are blunt ends or 5'overhangs (avoid 3'overhangs appear), the recommended template amount for each reaction is 1 μg;
b. Use the PCR product of the T7 promoter or the synthesized DNA fragment as a template: when PCR amplifying the template, add the T7 promoter (5'-TAATACGACTCACTATAGGG-3') to the 5'end of the non-coding strand primer. PCR products can be used directly as transcription templates without purification, but more RNA will be produced after purification. The recommended template amount for each reaction is about 0.5 μg.
2. Transcription reaction: According to the recommended reaction system in the table, add various reagents to the clean EP tube, mix well, and react at 37°C for 2 hours. (If synthesizing RNA less than 300 nt, it is recommended to extend the reaction time to 4 h or longer).
5×T7 Transcription Reaction Buffer
25 mM NTP Mix
T7 RNA Transcription Enzyme Mix
Nuclease Free Water
To 20 μL
3. After the reaction is completed, add 1 μL DNase I to the system, and react at 37°C for 15 minutes to digest the transcribed DNA template.
4. The synthesized RNA can be used in downstream experiments after electrophoresis analysis and purification.
5. Quantification and detection of transcription products: The concentration of RNA can be determined by ultraviolet absorption method (free nucleotides will affect the accuracy of quantification, RNA products need to be purified); electrophoresis detection is recommended to use 1% formaldehyde agarose denaturing gel detection, electrophoresis solution is 1×MOPS Buffer (10×MOPS Buffer: 0.4 M MOPS, pH 7.0, 0.1 M Sodium Acetate, 10 mM EDTA). Gel preparation method: Weigh 0.5 g of agarose into 36 mL RNase-Free Water, heat to melt, and add 5 mL 10×MOPS Buffer. When the solution is cooled to not hot, add 9 mL of formaldehyde solution (37%), mix well and pour the glue. During electrophoresis detection, mix an appropriate amount of RNA with RNA Loading Buffer, incubate at 70°C for 10 minutes, then ice bath for 2 minutes, and spot all samples. After the electrophoresis, stain with EB or SerRed (G3606) for observation.
1. The surface of human skin is rich in RNase. Please wear experimental gloves and masks when experimenting. The experimental consumables are sterile and enzyme-free to prevent RNase contamination.
2. If you need to prepare labeled RNA, please replace the NTP Mix in the kit.
3. The transcription length of the control template in the kit is 500 nt.
4. For your safety and health, please wear lab coats and disposable gloves for operation.