商品编号


M2080S-400 units



品牌


纽英伦(NEB)



公司


New England Biolabs



公司分类


RNA Reagents



Concentration

10,000 units/ml


Size

400 units

商品信息

Description:
Based on the Vaccinia virus Capping Enzyme (VCE), the Vaccinia Capping System provides the?necessary components to add 7-methylguanylate cap structures (Cap 0) to the 5?end of RNA (1). In eukaryotes, these terminal cap structures are involved in st
ABI
lization (2), transport (3), and translation (4) of mRNAs. Enzymatic production of capped RNA is an easy way to improve the st
ABI
lity and translational competence of RNA used for
in?vitro
translation, transfection, and microinjection. Alternatively, use of labeled GTP in a reaction provides a convenient way to label any RNA containing a 5? terminal triphosphate.
This single enzyme is composed of two subunits (D1 and D12) and has three enzymatic activities (RNA triphosphatase and guanylyltransferase by the D1 subunit and guanine methyltransferase by the D12 subunit); all necessary for addition of a complete Cap 0 structure, m7Gppp5?N (5,6).
In vitro
transcripts can be capped in less than one hour in the presence of the capping enzyme, reaction buffer, GTP, and the methyl donor, SAM. Capping is nearly 100% efficient and all capped structures are added in the proper orientation, unlike co-transcriptional addition of some cap analogs (7).
Product Source
An?
E. coli
?strain that carries the genes for the Vaccinia (WR) capping enzyme.
Reagents Supplied
The following reagents are supplied with this product:
Store at (°C)
Concentration
Capping Buffer
10X
S-adenosylmethionine (SAM)
-20
32 mM
GTP
-20
10 mM
Notes:
(read prior to setting up reaction)RNA used for capping reactions should be purified prior to use and s
USP
ended in nuclease-free water. EDTA should not be present and the solution should be free of salts.While RNase Inhibitor is not required, many users prefer to use it to enhance the st
ABI
lity of their RNA in solution. If this is desired, 0.5 ?l of a standard RNase inhibitor prep (such as RNase Inhibitor, Murine,
NEB
#M0314) can be added at the time of reaction set-up. The additional volume can be subtracted from the amount of H2O used in step 1 of both the capping and the labeling protocols.Heating the solution of RNA prior to incubation with the Vaccinia Capping Enzyme removes secondary structure on the 5? end of the transcript. Extend time to 10 minutes for transcripts with known highly structured 5? ends.SAM is unstable at pH 7–8, 37°C and should be mixed fresh prior to starting the reaction. We recommend determining how many reactions will be performed and diluting an aliquot of the 32 mM stock to 2 mM just prior to setting up the reactions. This "working stock" should be kept on ice to prevent degradation of SAM.For transcripts with known structured 5? ends, the reaction time can be extended to 60 minutes to improve capping efficiency.For labeling the 5? end, the total GTP concentration should be around 1–3X the molar concentration of mRNA in the reaction. The 10 mM stock can be diluted and a "spike" of hot GTP added to make the GTP mix.