T7 High Efficiency Transcription Kit RNA polymerase by TransGen Biotech

Product Overview

Description

T7 High Efficiency Transcription Kit

 

T7 High Efficiency Transcription Kit is designed for RNA synthesis by T7 RNA Polymerase with supercoiled or linearized DNA templates. This kit is suitable for production of large amount of RNA (up to 150 μg/per reaction) form DNA template size of 0.1-5 kb. Synthesized RNA can be used for in vitro translation, RNase protection assays, RNA splicing and hybridization based blots.

 

Cat. No.	JT101
Specification	JT101 -01	20ul (25rxns)
Storage:	at -20°C for two years
Application	In vitro site-directed mutagenesis, digestion of methylated plasmid template.
Notes	RNase contamination should be avoided.
	Transcript produced from the control template is 2 kb. Please refer to corresponding section above for transcription, purification, quantification and analysis.

 

Kit Contents

Component	JT111-01
T7 Transcription Enzyme Mix	50 μl
5×T7 Transcription Reaction Buffer	100 μl
10 mM NTP Mix	100 μl
DNase I (1 unit/μl)	25 μl
500 mM EDTA (pH 8.0)	25 μl
RNase-free Water	500 μl
Control Transcription Template (0.5 μg/μl)	10 μl

 

Protocol

RNA Synthesis
Principle of Transcription T7 RNA Polymerase

 

Template Preparation
• Supercoiled plasmid DNA
Supercoiled plasmid DNA should contain a T7 promoter and an effective terminator. Termination efficiency terminator varies with teminator. The following sequence has strong termination efficiency.

 

 

• Linearized DNA
Linearized DNA can be generated by restriction enzyme digested plasmid or use PCR product as template. When use linearized templates avoided 3’-overhangs, we recommend to use restriction enzymes that
produce 5’-overhangs or blunt ends. If 3’-overhangs cannot be avoided, template should be treated with T4 DNA polymerase to generate blunt ends. After digestion, template DNA should be purified.

 

Transcription

• Add following components

Components	Volume
Template	1 μg
5×Transcription Reaction Buffer	4 μl
10 mM NTP Mix	4 μl
Transcription Enzyme Mix	2 μl
RNase-free water	to 20 μl

 

• Mix thoroughly and incubate at 37 o C for 2 hours.
• Add 1 μl DNase I, incubate at 37 o C for 15 minutes. Then add 1 μl of 500 mM EDTA (pH 8.0) to terminate reaction (immediately proceed with following purification step after termination).

 

Purification of Synthesized RNA

Please refer to EasyPure ® RNA Purification Kit.

 

Quantification and Analysis of synthesized RNA

• RNA concentration can be determined by ultraviolet light spectrophotometer.
• Transcripts at 0.1-1 kb can be run on denaturing gel (6% acrylamide, 7 M urea). Electrophoretic buffer is

  1×TBE Buffer. 10×TBE Buffer: 0.9 M Tris Base, 0.9 M Boric Acid, 20 mM EDTA. Gel Formula: for each

  10 ml gel, add 4.2 g urea, 4.4 ml water, 1.5 ml of 40% ( acrylamide: methylene bis acrylamide =19:1

  (w/w) acrylamide, 1 ml 10×TBE, 100 ul of 10% AP, 10 μl TEMED. AP and TEMED should be added

  after urea completely dissolves.
• Transcripts at 0.5-5 kb can also be run on 1% formaldehyde denaturing gel. Electrophoretic buffer is

  1×MOPS Buffer. 10×MOPS Buffer: 0.4 M MOPS (pH 7.0), 0.1 M Sodium Acetate, 10 mM EDTA. Gel

  Formula: for each 100 ml gel, add 1 g agarose into 72 ml RNase-free water, dissolve it by heating, then

  add 10 ml of 10×MOPS Buffer. Cool the solution until 50-60oC, add 18 ml formaldehyde (37%), mix

  thoroughly and pool the gel.
• To do electrophoresis analysis, dilute 0.2-1 μg RNA with RNase-free water into 5 μl, add same volume of

  2×RNA Loading Buffer and mix thoroughly, incubate at 70 o C for 10 minutes and followed by incubation

  on ice for 2 minutes, load samples on the gel. After electrophoresis, stain by EB or other nucleic acid dye

  for observation. RNA Marker is processed with the same method as RNA sample in this step for

   electrophoresis analysis (or referring to supplier’s manual).

 

CITATIONS

Li M, et al. 2013. Stability and iron oxidation properties of a novel homopolymeric plant ferritin from adzuki bean seeds: A comparative analysis with recombinant soybean seed H-1 chain ferritin. 1830(4):2946-53. Biochim Biophys Acta. IF=3.848. PMID: 23313843.