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mRNA Vaccinia Capping Enzyme, Animal Free, Antibiotic Free

Product SKU

Specifications

Price

10614ES84

2000 U

$658.52

10614ES92

10000 U

$2752.18

10614ES94

20000 U

$4604.98

10614ES96

100000 U

$18423.78

Product Description

Eukaryotes mRNA forms a special structure at the 5'end after transcription, which is the cap structure. The cap structure plays an important role in the stability, transportation and translation of mRNA. Vaccinia virus capping enzyme is an effective enzyme that catalyzes the formation of the cap structure. It’s composed of two subunits D1 and D12. It also has RNA triphosphatase activity, guanylate acyltransferase activity and guanine methyltransferase activity, could connect the 7-methylguanine cap structure (m7Gppp) to the 5'end of the RNA (m7Gppp5'N). Vaccinia virus capping enzyme can cap the RNA at correct direction within one hour when present at suitable concentration of capping buffer (Capping buffer), guanosine triphosphate (GTP), S-adenosylmethionine (SAM), etc..

This product is produced in accordance with GMP process requirements and provided in a sterile liquid form, used for in vivo/in vitro pre-translation mRNA capping reaction or mRNA 5'end labeling reaction.

Product Nature

Source

E. coli with vaccinia virus capping enzyme gene

Optimum Temperature

37℃

Host nucleic acid residue

<1fg /U

Host protein residue

<50 ppm

Pathogen(HBV/ HCV/HIV)

Negative

Endotoxin

<0.05 EU/1000 U

Mycoplasma detection

Negative

Sterility

Negative

Storage Buffer

20 mM Tris-HCl pH 8.0,100 mM NaCl,1 mM DTT,0.1 mM EDTA,0.1% Triton X-100,50% glycerin

Unit Definition

1 unit: The amount of enzyme required to incorporate 10 pmol GTP (α- 32P) into a transcript with 80 nucleotides (80 nt) at 37℃ within 1 hour.

Note: For specific product quality inspection data and more indicators, please check the batch quality inspection report.

Contents

Contents No.

Name

10614ES84(2000 U)

10614ES92 (10000 U)

10614ES94 (20000 U)

10614ES96(100000 U)

10614-A

10×Capping Buffer

500 μL

1.25 mL×2

1.25 mL×4

25mL

10614-B

Vaccinia Capping Enzyme(10 U/μL)

200 μL

1 mL

1 mL×2

10 mL

Shipping and Storage

Shipped on wet ice, store at -20℃ for one year.

Notes:

For your safety and health, please wear lab clothes and disposable gloves.

The extracted RNA needs to be purified and resuspended in nuclease-free water;

The RNA solution needs to be heated before adding the enzyme to remove the secondary structure at the 5'end;

For RNA with a known 5'end structure, the reaction time can be extended to 60 minutes to improve the capping efficiency;

In the 5'end labeling reaction system, the GTP stock solution should be diluted to 1-3 times of the mRNA molar concentration in the reaction system.

Experimental methods

1. Capping reaction (20 μL reaction system)

This step is suitable for capping reaction of 10 μg RNA (≥ 100 nt), and can be amplified according to experimental needs.

1) Take 10 μg RNA to a 1.5 mL centrifuge tube and dilute to 15 μL with nuclease-free water;

2) Heat at 65°C for 5 minutes;

3) Take out the centrifuge tube and place it on ice for 5 minutes;

4) Add the following components in sequence:

Components

Volume

Denatured RNA

15 μL

10×Capping Buffer

2.0 μL

GTP(10 mM)

1.0 μL

SAM(2 mM)

1.0 μL

Vaccinia Capping Enzyme(10 U/μL)

1.0 μL

【Note】10×Capping Buffer:0.5 M Tris-HCl,pH 8.0;50 mM KCl,10 mM MgCl2,10 mM DTT。

5)Incubate at 37°C for 30 minutes;

6)RNA capping is completed, subsequent experiments can be performed。

2. 5'end labeling reaction(20 μL reaction system)

This step is suitable for RNA labeling with triphosphate at the 5'end, and can be amplified according to experimental needs. The labeling efficiency is affected by the molar concentration ratio of RNA to GTP in the reaction system and the content of GTP in the RNA sample.

1) Take appropriate amount of RNA to a 1.5 mL centrifuge tube and dilute to 15 μL with nuclease-free water;

2) Heat at 65°C for 5 minutes;

3) Take out the centrifuge tube and place it on ice for 5 minutes;

4) Add the following components in sequence::

Components

Volume

Denatured RNA

14.0 μL

10×Capping Buffer

2.0 μL

GTP mix

2.0 μL

SAM(2 mM)

1.0 μL

Vaccinia Capping Enzyme(10 U/μL)

1.0 μL

【Note】GTP mix contains GTP and a small amount of markers. For the concentration of GTP, refer to Note 5).

5) Incubate at 37°C for 30 minutes;

6) The 5'end of RNA is labeled, and subsequent experiments can be performed.

15221439726

15221439726