The T7 Quick High Yield RNA Transcription Kit is a kit that uses T7 RNA polymerase to synthesize large amounts of RNA in vitro, with DNA containing the T7 promoter as the template. It is suitable for both long and short transcripts. The T7 Enzyme Mix provided in this product is pre-mixed with RNase inhibitor and inorganic pyrophosphatase, while DNase I, RNase-free is used to remove template DNA after the transcription reaction. Using this product with 1μg of linearized double-stranded DNA template can yield at least 150μg of RNA. The RNA synthesized by transcription can be used in various downstream applications, such as in vitro translation, RNA structure and function research, RNase protection, probe hybridization, and RNA interference.
Product Components and Storage Conditions
| SKU | Component
| 50T
| Storage
|
T1492421A
| T7 Enzyme Mix
| 100 μl
| -20℃
| T1492421
B
| 10× T7 RNA Pol Buffer
| 100 μl
| -20℃
|
T1492421C
| ATP (100mM)
| 100 μl
| -20℃
|
T1492421D
| UTP (100mM)
| 100 μl
| -20℃
|
T1492421E
| GTP (100mM)
| 100 μl
| -20℃
|
T1492421F
| CTP (100mM)
| 100 μl
| -20℃
|
T1492421G
| Control Template (0.5 μg/μl)
| 10 μl
| -20℃
|
T1492421H
| DNase I, RNase-free (1 U/μl)
| 100 μl
| -20℃
| T1492421I
| Nuclease-Free Water
| 1 ml
| -20℃ |
|
Transcription Protocol
Figure 1. RNA Transcription Protocol for Non-Cotranscriptional T7 Promoter
Usage Instructions
1. DNA Template Preparation
Linearized plasmids containing the T7 promoter or PCR amplification products can both be used as templates for in vitro transcription. The template can be dissolved in TE buffer or Nuclease-Free Water. T7 promoter sequence: TAATACGACTCACTATAN*
Note: N* is the first base of RNA transcription, which is generally G. If cotranscription is performed, it is determined by the cap analog.
(1) Plasmid Template
Insert the target DNA into a plasmid vector containing the T7 promoter, then treat it with restriction enzymes. After complete linearization, purify the plasmid.
Note 1: Since terminators cannot ensure 100% termination of transcription, circular plasmids will transcribe RNA products of different lengths. To obtain RNA of a specific length, the plasmid must be completely linearized.
Note 2: The restriction site of the restriction enzyme selected for plasmid linearization needs to be adjacent to the downstream of the coding strand and must not have a recognition site within the coding strand. The selected restriction enzyme should be capable of generating 5' overhangs or blunt ends.
Note 3: To avoid the impact of proteins, salts, and other substances on the transcription system, it is recommended to purify the linearized plasmid before using it as a template for in vitro transcription.
Note 4: Residual RNase A introduced during plasmid DNA extraction will significantly affect the quality of transcribed RNA. It is recommended to use high-purity RNase-free templates with an A260/A280 ratio of 1.8–2.0.
(2) PCR Product Template
PCR products carrying the T7 promoter can be used as templates for in vitro transcription. First, add the T7 promoter sequence to the 5' end of the upstream primer of the coding strand. Then, amplify the DNA template containing the T7 promoter under the action of a high-fidelity enzyme, followed by transcription. PCR products can be directly used as templates without purification, but the RNA yield will be higher after purification.
Note 1: When using PCR products as templates, it is necessary to confirm the specificity and concentration of the products by electrophoresis. It is recommended to add 2–5 μl of PCR products to a 20 μl reaction system.
Note 2: To obtain more high-quality RNA, it is recommended to purify the PCR products before using them as templates for in vitro transcription.
2. In Vitro Transcription
(1) Select one of the three reaction systems below for reaction solution sampling according to the type of product required.
① Standard In Vitro Transcription System
Prepare the following reaction system on ice:
Reagent
| Volume
| Final Concentration
| 10× T7 RNA Pol Buffer
| 2 μl
| 1×
| T7 Enzyme Mix
| 2 μl
| - | Template DNA
| 1 μg
| 50 ng/μl
| ATP/CTP/GTP/UTP (100 mM each)ᵃ’ᵇ
| 2 μl each
| 10 mM each
| Nuclease-Free Water
| up to 20 μl
| - |
|
a. It is recommended to add Nuclease-Free Water first, followed by ATP/CTP/GTP/UTP.
b. Modified NTPs with the same molar concentration can be used to replace the corresponding unmodified NTPs. For modified UTPs, reference can be made to the use of N¹-Methyl-Pseudo-UTP (100 mM) (Catalog No.: N464730) and Pseudo-UTP (100 mM) (Catalog No.: P463387).
② Capped RNA Cotranscription System
Prepare the following reaction system on ice:
Reagent
| Volume
| Final Concentration
| 10× T7 RNA Pol Buffer
| 2 μl
| 1×
| T7 Enzyme Mix
| 2 μl
| - | Template DNA
| 1 μg
| 50 ng/μl
| ATP/CTP/GTP/UTP (100 mM each)
| 2 μl each
| 10 mM each
| Cap1 Analogue (100 mM)ᶜ
| 1.6 μl
| 8 mM
| Nuclease-Free Water
| up to 20 μl
| - |
|
c. The molar concentration ratio of the cap analog to each NTP should be 4:5. This molar ratio is applicable to the CleanCap series of cap analogs. For the cap analog, reference can be made to the use of Cap1 analogue AG. If cap analogs with other structures are used, please set a reasonable ratio between the cap analog and GTP according to the cap analog's instruction manual. The ratio can be adjusted based on the capping efficiency, but it is advisable to control the sum of their final concentrations at 10 mM.
③ Non-Radioactive Labeled RNA In Vitro Transcription System
Reagent
| Volume
| Final Concentration
| 10× T7 RNA Pol Buffer
| 2 μl
| 1×
| T7 Enzyme Mix
| 2 μl
| - | Template DNA
| 1 μg
| 50 ng/μl
| ATP/CTP/GTP (100 mM each)
| 2 μl each
| 10 mM each
| UTP (100 mM)
| 1.5 μl
| 7.5 mM
| Modified UTP (50 mM)ᵈ
| 1 μl
| 2.5 mM
| Nuclease-Free Water
| up to 20 μl
| - |
|
d. This system is suitable for biotin-modified UTP, fluorescein-modified UTP, digoxin-modified UTP, or aminoallyl-modified UTP. The transcription yield using modified UTP will be lower than that using unmodified UTP.
Note 1: The transcription efficiency varies significantly among different template sequences. For initial experiments, follow the recommended addition amount first, then optimize the optimal system through exploration. The template amount can be adjusted within the range of 0.5 μg to 2 μg.
(2) After thorough mixing and brief centrifugation, incubate at 37°C for 2 hours. If the length of the transcription product is < 100 nt, the reaction time can be extended to 3–16 hours.
(3) After the reaction is completed, add DNase I, RNase-free to the product at a ratio of 2 μl per μg of Template DNA, and incubate at 37°C for 15 minutes to remove the DNA template.
(4) It is recommended to purify the transcribed RNA using magnetic beads or columns; phenol/chloroform or lithium chloride purification can also be used. The purified RNA can be used for downstream experiments or stored at -80°C for future use.
3. Control Template Transcription (Not Included in T Package)
The control template is a linear DNA fragment containing the T7 promoter, and the transcription product is approximately 4000 nt in size. In the recommended standard in vitro transcription reaction system, 1 μg of control template DNA can yield at least 150 μg of RNA when reacted at 37°C for 2 hours.
4. Product Purification
After transcription, the RNA can be purified using magnetic bead-based purification, column purification, phenol/chloroform purification, or lithium chloride precipitation to remove proteins and free nucleotides. The purified RNA can be used for downstream experiments after electrophoresis detection or stored at -80°C.
(1) Magnetic Bead Purification Method
Perform the purification operation according to the magnetic bead instruction manual.
(2) Column Purification Method
Before purification, add 80 μl of Nuclease-Free Water to dilute the product to 100 μl, then perform the purification operation according to the purification column instruction manual.
(3) Phenol/Chloroform Purification Method
① To 20 μl of the reaction mixture, add 115 μl of Nuclease-Free Water and 15 μl of 3M sodium acetate (pH 5.2), and mix thoroughly.
② Add an equal volume (150 μl) of phenol/chloroform (1:1) mixture for extraction once. Centrifuge at room temperature at maximum speed (≥12000 rpm) for 5 minutes, then transfer the upper aqueous phase to a new RNase-free EP tube.
Note: Do not aspirate the middle layer when transferring the supernatant.
③ Add an equal volume of chloroform for extraction twice more. Collect the supernatant and transfer it to a new RNase-free EP tube.
④ Add 2 volumes of absolute ethanol to precipitate the RNA. After mixing thoroughly, place it at -20°C for at least 30 minutes. Centrifuge at 4°C at maximum speed (≥12000 rpm) for 15 minutes, then collect the precipitate.
⑤ Add 500 μl of ice-precooled 70% ethanol to wash the RNA precipitate. Centrifuge at 4°C at maximum speed (≥12000 rpm) for 5 minutes, then collect the precipitate.
⑥ Dissolve the RNA precipitate with 20 μl of Nuclease-Free Water. Store the purified RNA solution at -80°C.
(4) Lithium Chloride Precipitation Method
For the lithium chloride precipitation method, the RNA must have a length of at least 100 nt and a concentration of no less than 100 ng/μl.
① To 20 μl of the reaction mixture, add 30 μl of Nuclease-Free Water and 30 μl of 7.5 M lithium chloride.
② After mixing thoroughly, place it at -20°C for at least 30 minutes. Centrifuge at 4°C at maximum speed (≥12000 rpm) for 15 minutes, then collect the precipitate.
③ Add 500 μl of ice-precooled 70% ethanol to wash the RNA precipitate. Centrifuge at 4°C at maximum speed (≥12000 rpm) for 5 minutes, then collect the precipitate.
④ Dissolve the RNA precipitate with 20 μl of Nuclease-Free Water. Store the purified RNA solution at -80°C.
5. RNA Quantification
(1) UV Absorption Method: Free NTPs and other substances will seriously affect the accuracy of quantification. Please purify the RNA first before using this method.
(2) Dye-Based Method: RNA-specific fluorescent dyes or related kits can be used for RNA quantification. This method allows quantification of RNA in both purified and unpurified reaction products.
6. RNA Detection
(1) Gel Electrophoresis Method
To evaluate the length and quality of transcripts, the transcription products should be subjected to electrophoresis detection using an appropriate non-denaturing or denaturing agarose or polyacrylamide gel. Denaturing electrophoresis can reduce the formation of RNA secondary structures, and usually, RNA migrates as a single band of the correct size.
Transcript Length
| Recommended Electrophoretic Gels
| >500 nt
| 1% Agarose Gel
| 100~500 nt
| 2% Agarose Gel or 4–5% Urea-Denaturing Polyacrylamide Gel
| 50~100 nt
| 10%–15% Urea-Denaturing Polyacrylamide Gel
| <50 nt
| 20% Urea-Denaturing Polyacrylamide Gel |
|
Note 1: Both the electrophoresis buffer and gel should be prepared and used immediately. When performing urea-denaturing polyacrylamide gel electrophoresis, it is recommended to use 0.5× TBE (Catalog No.: T397904) as the electrophoresis buffer.
Note 2: The transcribed RNA can be diluted with Nuclease-Free Water before electrophoresis, and the recommended loading volume for electrophoresis is 0.05–1 μg.
Note 3: After adding the RNA Loading Buffer to the sample, the sample can be treated at 65°C for 5–10 minutes to reduce the formation of RNA secondary structures.
Note 4: Safe Red nucleic acid dye (Catalog No.: S397951) can be used to visualize RNA electrophoresis bands. For polyacrylamide gels, the soaking method is recommended for band visualization.
(2) Capillary Electrophoresis Method
Capillary electrophoresis can digitally and accurately evaluate the integrity, purity, or degradation degree of RNA samples. Compared with the gel method, this method requires a smaller amount of RNA sample and has higher sensitivity.
Common Questions
Problem Description
| Reason
| Example sentence
| Low yield of transcription products
| There may be components in the template that inhibit the reaction, or the template purity is poor, or the template concentration is incorrect.
| If the yield of the control group is normal, please repurify the template and confirm the template concentration as well as its specificity. If the yield of the control group is low, please consult the technical support of our company.
| Low yield of short-fragment transcription products
| A short transcription initiation fragment may inhibit the reaction.
| When the transcription product is 100 nt, it is recommended to extend the reaction time or increase the amount of template to 2 μg.
| The RNA product fragments are smaller than expected
| The template sequence may contain a termination sequence similar to that of T7 RNA polymerase.
| It is recommended that lowering the reaction temperature to 30°C may increase the proportion of full-length transcripts, but the yield will decrease.
|
| The template may have a high GC content or form secondary structures.
| It is recommended to increase the reaction temperature to 42°C or use single-stranded binding proteins to improve the yield and length (of the transcription products).
| The RNA product fragments are larger than expected
| The plasmid template DNA may be incompletely digested by restriction enzymes.
| It is recommended to continue the restriction enzyme digestion of the plasmid template DNA or re-optimize the enzyme digestion reaction system for plasmid template DNA preparation.
|
| The RNA may have stronger secondary structures and thus not be completely denatured.
| Increase the concentration of the denaturant, or raise the sample denaturation temperature to 70°C after adding the RNA Loading Buffer.
| Electrophoretic tailing
| RNase contamination may occur during the experimental process.
| It is recommended to have a dedicated operating area for transcription reactions, as well as specialized RNA reagents and pipettes; wear a mask and gloves during the experiment, and replace gloves frequently; use RNase-free RNA consumables; keep the reagent lids closed when the reagents are not in use; ensure that the test tubes are tightly sealed during the reaction process.
|
| The template may be contaminated with RNase.
| RNase contamination in the template may also result in RNA product fragments being smaller than expected. It is recommended to repurify the template DNA and then dissolve it in Nuclease-Free Water or DEPC-treated water. |
|
