| Field | Specification |
|---|---|
| Product Type | |
| Shipping | |
| Storage |
Description
The kit utilizes T7 RNA Polymerase (Low dsRNA) and high-affinity LZcap AG Cap1 analog to produce mRNAs with significantly reduced double-stranded RNA (dsRNA) content and enhanced expression. Single-stranded RNAs are synthesized using linear double-stranded DNA containing a T7 promoter sequence as the template. Modified nucleotides, such as biotin or dye-labeled nucleotides, can be incorporated during transcription to generate labeled RNA. The kit supports the synthesis of both long and short transcripts, yielding 100–200 μg of RNA from 1 μg of DNA template. Substituting UTP with N1-Me-Pseudo UTP reduces RNA immunogenicity. The resulting RNA is suitable for downstream applications, including RNA structure and function studies, RNase protection, probe hybridization, RNAi, microinjection, and in vitro translation.
Components
|
Components No. |
Name |
10635ES10 (10 T) |
10635ES50 (50 T) |
10635ES60 (100 T) |
10635ES70 (500 T) |
|
10635-A |
T7 RNA Polymerase Mix |
20 μL |
100 μL |
200 μL |
1 mL |
|
10635-B |
10×Transcription Buffer |
20 μL |
100 μL |
200 μL |
1 mL |
|
10635-C |
ATP（100mM） |
20 μL |
100 μL |
200 μL |
1 mL |
|
10635-D |
CTP（100mM） |
20 μL |
100 μL |
200 μL |
1 mL |
|
10635-E |
GTP（100mM） |
20 μL |
100 μL |
200 μL |
1 mL |
|
10635-F |
N1-Me-Pseudo UTP（100mM） |
20 μL |
100 μL |
200 μL |
1 mL |
|
10635-G |
Control DNA Template（500ng/μL） |
5 μL |
10 μL |
20 μL |
100 μL |
|
10635-H |
DNase I (2 U/μL) |
10 μL |
50 μL |
100 μL |
500 μL |
|
10635-I |
Cap1 Analog (100 mM) |
20 μL |
100 μL |
200 μL |
1 mL |
|
10635-J |
Lithium chloride solution |
300 μL |
1.5 mL |
3 mL |
15 mL |
|
10635-K |
RNase-free ddH2O |
500 μL |
2.5 mL |
5 mL |
25 mL |
Note: The T7 RNA Polymerase Mix includes T7 RNA polymerase (Low dsRNA, Cat: 10628), Murine RNase inhibitor (Cat: 10621) and Pyrophosphatase Inorganic (Cat: 10620); The Cap1 Analog is LZcap AG (Cat: 10684). The catalog number of recommended UTP and Pseudo UTP is 10654 and 10656, respectively.
Features
- Complete Kit Components: Includes all essential reagents for one-step co-transcriptional capping, such as NTPs, T7 Enzyme Mix, cap analog, DNase I, and LiCl.
- Simplified Workflow: Utilizes a co-transcriptional capping process, minimizing the number of components introduced and streamlining the overall procedure for ease of use.
- High Yield: Generates 150–200 µg of Cap 1 mRNA from just 1 µg of DNA template.
- Reduced Immunogenicity: Incorporates modified N1-Me-pUTP and a low dsRNA T7 RNA polymerase to minimize unintended innate immune responses.
- High Capping Efficiency: Achieves capping efficiency exceeding 99%.
Figure
1. Broad Applicability:
Consistently delivers a high yield of 9 mg/mL with capping efficiency up to 100% in a 20 µL reaction system using 1 µg of plasmid DNA, regardless of the length of the target transcript.
|
mRNA Category |
Yield(mg/mL) |
mRNA Integrity(%) |
Capping Efficiency(%) |
|
eGFP |
11.2 |
93.5 |
100% |
|
OVA |
10.2 |
94.3 |
100% |
|
mcherry |
9.1 |
90.3 |
100% |
|
Luciferase |
9.3 |
90.1 |
100% |
|
Cas 9 |
9 |
88.7 |
100% |
Table 1. Broad applicability of the Co-transcription system
|
|
Cap analog |
Cap Concn. (mM) |
Yields (mg/mL) |
Integrity (%) |
Capping efficiency by MS (%) |
|
1 |
Cap AG (3'OMe-7mG) |
10 |
12.69 |
80.6 |
100 |
|
2 |
LZCap AG |
10 |
12.11 |
82.8 |
100 |
|
3 |
LZCap AG |
7.5 |
11.59 |
82.2 |
100 |
|
4 |
LZCap AG |
5 |
11.16 |
81.6 |
100 |
|
5 |
LZCap AG |
2.5 |
10.75 |
81.4 |
100 |
Figure 1. Co-transcription analysis of the input of LZcap AG. IVT materials include Hieff™ T7 RNA Polymerase, Template: Cas9 (4K); T7 Reaction Buffer 2; Tris NTP (including N1-Me-Pseudo UTP Tris Solution). IVT time: 3 hrs.
|
Fragment Length |
T7 RNA pol |
Yield (mg/mL) |
Integrity (%) |
dsRNA Content (ng/ug of RNA) |
|
4K mRNA |
Regular T7 Kit |
12.4 |
88.3 |
0.4273 |
|
Low dsRNA T7 Kit |
12.1 |
89.9 |
0.0129 |
|
|
9K mRNA |
Regular T7 Kit |
9.5 |
81.9 |
2.9180 |
|
Low dsRNA T7 Kit |
9.0 |
82.0 |
0.0379 |
Table 2. dsRNA content analysis of regular T7 Kit and Low dsRNA T7 Kit.
Storage
This product should be stored at -25 ~-15℃ for one year.
Notes:
1. Be careful not to mix RNase in the reaction system.
2. Experiment equipment (such as: pipette tip, product tube, etc.) should strictly use RNase-free products.
3. For your safety and health, please wear lab coats and disposable gloves.
4. For research use only!
Document
Safety Data Sheet
10635_MSDS
Manual
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