T7 High Yield Transcription Kit

T7 High Yield Transcription Kit

Cat. No.: SB-G3021-50T
Size: 50T


Description

This kit uses T7 RNA Polymerase to transcribe and synthesize RNA in vitro with linearized plasmid DNA containing T7 promoter, PCR product or synthetic DNA as template and NTP as substrate. This kit optimizes the RNA transcription reaction system in vitro, which can obtain a large number of RNA molecules simply and quickly. If modified nucleotides are added to the substrate during transcription, biotin or dye-labeled RNA can be prepared. This kit is mainly used for in vitro translation, RNase protection experiments, hybridization probe labeling, RNA shearing and other biological experiments. In the reaction system, 1 μg of template input can produce more than 100 μg of RNA, which is suitable for the preparation of RNA of various lengths.

 

Storage and Handling Conditions

Transport with wet ice; Store at -20℃, valid for 12 months.


Component
 

Component Number	Component	G3021-50T
G3021-1	T7 RNA Transcription Enzyme Mix	200 μL
G3021-2	5×T7 Transcription Reaction Buffer	250 μL
G3021-3	25 mM NTP Mix	100 μL
G3021-4	DNase I	50 μL
G3021-5	Nuclease Free Water	1 mL
G3021-6	Control Template (0.5 μg/μL)	10 μL
Product Manual

Assay Protocol

1. Preparation for template :

A. Plasmid from the T7 promoter as template: to obtain RNA of a specific length, the plasmid template must be fully linearized (purified as template), and the linearized plasmid ensures that the double strand is flat or 5 'protruding (avoid 3' protruding). The recommended amount of template for each reaction is 1 μg;

B. The PCR product of the T7 promoter or the synthesized DNA fragment was used as the template: the T7 promoter (5’-TAATACGACTCACTATAGGG-3’) was added to the 5 'end of the primer of the non-coding strand during PCR amplification template. PCR products can be directly used as transcription templates without purification, but more RNA will be produced after purification. The recommended amount of template for each reaction is about 0.5 μg.

2. Transcription reaction: Add various reagents into a clean EP tube according to the reaction system recommended in the table, mix thoroughly and react at 37℃ for 2 h. (For RNA synthesis less than 300 nt, it is recommended to extend the reaction time to 4 h or longer).

 

Component	Volume
Template	0.5-1 μg
5×T7 Transcription Reaction Buffer	4 μL
25 mM NTP Mix	4 μL
T7 RNA Transcription Enzyme Mix	4 μL
Nuclease Free Water	To 20 μL

 

1. After the reaction, 1μl DNase I was added to the system, and the reaction was carried out at 37℃ for 15 min to digest the transcribed DNA template.

2. The RNA synthesized by transcription can be used in downstream experiments after electrophoresis analysis and purification.

3. Quantification and detection of transcription products: RNA concentration can be determined by UV absorption method (free nucleotides will affect the accuracy of quantification, and RNA products need to be purified); For electrophoresis detection, 1% formaldehyde agarose denaturing gel was recommended. The electrophoresis solution was 1×MOPS Buffer (10×MOPS Buffer: 0.4 M MOPS, pH 7.0, 0.1 M Sodium Acetate, 10 mM EDTA). Gel preparation method: 0.5 g agarose was weighed and added into 36 mL Rnase-free Water. After heating and melting, 5 mL of 10×MOPS Buffer was added. When the solution is cooled to not hot to handle, add 9 mL formaldehyde solution (37%), mix well and pour glue. For electrophoresis detection, an appropriate amount of RNA was mixed with RNA Loading Buffer, incubated at 70℃ for 10 min, followed by an ice bath for 2 min, and all samples were collected. After electrophoresis, EB or SerRed (G3606) staining was used to observe.

Note:

1. Human skin surface is rich in RNase. Please wear experimental gloves and masks during the experiment, and the experimental consumables are sterile and enzyme-free to prevent RNase contamination.

2. To prepare labeled RNA, please replace NTP Mix in the kit.

3. The transcription length of the control template in the kit was 500 nt.

4. For your safety and health, please wear a lab coat and disposable gloves when operating.