mRNA Cap2'-O-Methyltransferase

mRNA Cap 2´ -O-methyltransferase tau muab los ntawm ib hom E. coli recombinant uas nqa cov noob rau cov tshuaj tiv thaiv mRNA Cap 2 ´-O-Methyltransferase.Cov enzyme no ntxiv ib pab pawg methyl ntawm 2'-O txoj hauj lwm ntawm thawj nucleotide nyob ib sab ntawm lub hau qauv ntawm 5' kawg ntawm RNA.Lub enzyme siv S-adenosylmethionine (SAM) ua methyl pub rau methylate capped RNA (cap. -0) ua rau lub cap-1 qauv.

Cov qauv Cap1 tuaj yeem ua rau kom muaj txiaj ntsig zoo ntawm kev txhais lus, txhim kho kev qhia ntawm mRNA hauv kev hloov pauv thiab microinjection thwmsim.Qhov enzyme tshwj xeeb yuav tsum tau RNA nrog rau m7GpppN cap ua substrate.Nws tsis tuaj yeem siv RNA nrog pN, ppN, pppN lossis GpppN ntawm 5' kawg.Capped RNA tuaj yeem npaj tau los ntawm kev sau hauv vitro siv cap analog lossis los ntawm enzymatic capping siv Vaccinia Capping Enzyme.

Cheebtsam

mRNA Cap 2'-O-Methyltransferase (50U/μL)

10 × Capping Reaction Buffer

Cia

-25 ～-15 ℃ rau kev cia (Tsis txhob rov qab khov-thaw mus)

Cia tsis

20 mM Tris-HCl (pH 8.0, 25 ℃), 100 mM NaCl, 1 mM DTT, 0. 1 mM EDTA, 0. 1% Triton X- 100, 50% glycerol.

Unit txhais

Ib chav tsev yog txhais raws li tus nqi ntawm cov enzyme uas yuav tsum tau rau methylate 10 pmoles ntawm 80 nt capped RNA transcript hauv 1 teev ntawm 37 ° C.

Kev soj ntsuam zoo

Exonuclease:50U ntawm mRNA Cap 2 ´ -O-Methyltransferase nrog 1μg λ-Hind III digest DNA ntawm 37 ℃ rau 16 teev yields tsis degradation raws li txiav txim los ntawm agarose gel electrophoresis.

Endonuclease: 50 U ntawm mRNA Cap 2 ´ -O-Methyltransferase nrog 1 μg λDNA ntawm 37 ℃ rau 16 teev yields tsis degradation raws li txiav txim los ntawm agarose gel electrophoresis.

Nickase: 50U ntawm mRNA Cap 2 ´ -O-Methyltransferase nrog 1μg pBR322 ntawm 37 ℃ rau 16 teev yields tsis degradation raws li txiav txim los ntawm agarose gel electrophoresis.

RNase: 50U ntawm mRNA Cap 2 ´ -O-Methyltransferase nrog 1.6μg MS2 RNA rau 4 teev ntawm 37 ℃ yields tsis degradation raws li txiav txim los ntawm agarose gel electrophoresis.

E. coli DNA: 50U ntawm mRNA Cap 2 ´ -O-Methyltransferase yog tshuaj xyuas rau qhov muajE. coli genomic DNA siv TaqMan qPCR nrog primers tshwj xeeb rau covE. coli 16S rRNA chaw.CovE. coli genomic DNA contamination yog = 1E. coli genome.

Kab mob Endotoxin: LAL-test, raws li Suav Pharmacopoeia IV 2020 tsab, gel limit test method, general rule (1143).Cov kab mob endotoxin cov ntsiab lus yuav tsum yog = 10 EU / mg.

Cov tshuaj tiv thaiv kab mob thiab cov xwm txheej

1. Muab qhov tsim nyog ntawm Capped RNA thiab RNase-dawb H2O hauv 1.5 mL microfuge raj rau qhov kawg ntim ntawm 16.0 µL.

2. Tshav kub ntawm 65 ℃ rau 5 feeb ua raws li dej khov rau 5 feeb.

3. Ntxiv cov khoom nram qab no hauv qhov kev txiav txim tau teev tseg (rau methylation ntawm Capped RNA

tsawg dua 10

* 10 × Capping Reaction Buffer: 500 mM Tris-HCl (pH 8.0, 25 ℃), 50 mM KCl, 10 mM MgCl2, 10 mM DTT.

4. Incubate ntawm 37 ℃ rau 1 teev (2 teev ntawm incubation yog pom zoo rau lub hom phiaj fragment tsawg tshaj 200 nt).

Daim ntawv thov

Txhawm rau txhim kho mRNA kev qhia thaum lub sij hawm microinjection thiab transfection sim.

Cov lus qhia txog kev siv

1. Ua ntej cov tshuaj tiv thaiv, RNA yuav tsum tau ntxuav thiab yaj hauv cov dej tsis muaj nuclease, tag nrho cov kev daws teeb meem yuav tsum tsis muaj EDTA thiab ions.

2. Nws raug nquahu kom sov cov qauv RNA ntawm 65 ℃ rau 5 min ua ntej cov tshuaj tiv thaiv kom tshem tawm cov txheej txheem thib ob ntawm 5' kawg ntawm daim ntawv teev lus.Nws tuaj yeem txuas ntxiv mus rau 10 min rau complex 5 '-terminal qauv.