T7 High Yield Transcription Kit

 

Product Name	Cat.No.	Spec.
T7 High Yield Transcription Kit	G3021-50T	50 T

 

 

The T7 High Yield Transcription Kit is a complete system for the efficient synthesis of RNA for blot in situ hybridization probes,ribonuclease protection assays, post-transcriptional modifications including RNA splicing and polyadenylation, and for in vitro translation. T7 RNA polymerase is highly specific for its own promoter and will transcribe large amounts of RNA from DNA sequences (for example, plasmids, polymerase chain reaction (PCR) fragments, or Synthetic DNA) downstream of its promoter,

In the reaction system, 1 μg of template input can produce more than 100 μg of RNA, which is suitable for the preparation of various lengths of RNA.

 

 

 

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

 

 

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
Manual		One copy

 

 

1. Preparation for template :

a. Plasmid containing the T7 promoter : to obtain RNA of a specific length, the plasmid should be fully linearized and purified as template. Plasmid linealization with a restriction enzyme should produce a 5' overhang or a blunt end (avoid 3' protruding end). The recommended amount of template per reaction is ~1 μg;

b. The PCR product or the synthetic DNA: the 5 'end of the primer of the non-coding strand fusioned with the sequence from T7 promoter (5'-TAATACGACTCACTATAGGG-3') perform PCR amplification.. The PCR products can be directly used as transcription templates without purification, but RNA yield will be increase after purification. The recommended amount of template per reaction is ~0.5 μg.

2. In vitro translation reaction :

a. Add the following component into a sterile, nuclease-free tube on ice in the indicated order

b. Mix gently and centrifuge briefly.

c. Incubate at 37℃ for 2 h. (Adjust the reaction time depending on the target length and the desired RNA yield. For example , RNA less than 300 nt, it is recommended to incubate for 4 h or longer).

Component	Amount	Final conc.
Template	0.5-1 μg	25-50 µg/mL
5×T7 Transcription Reaction Buffer	4 μL	1×
25 mM NTP Mix	2 μL	2.5 mM
T7 RNA Transcription Enzyme Mix	4 μL
Nuclease-Free Water	Up to 20 μL	--

3. DNase I Treatment

After the reaction, add 1 μL of DNase I, mix briefly and incubate at 37℃ for 15 minutes.

4. Quantification of transcription products

a. Gel electrophoresis analysis

1% denaturing formaldehyde agarose gel is recommended.

b. UV absorption method

The concentration of RNA products determined by UV absorption equipments aftre purificaton (free nucleotides will affect the accuracy of quantification).

 

 

1. Please wear appropriate gloves and masks and use disposable RNase-free consumables to prevent RNase contamination.

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

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

4. For your safty and health,please wear safety glasses, gloves, or protective clothing.

 

For Research Use Only!