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Isithombe Esifakiwe se-T7 RNA Polymerase HCP1011A
  • I-T7 RNA Polymerase HCP1011A

I-T7 RNA Polymerase


Inombolo yekati:HCP1011A

Iphakheji: 100μL/1mL/10mL/100mL

I-T7 RNA Polymerase iyi-RNA polymerase encike ku-DNA evela ku-T7 phage

Incazelo Yomkhiqizo

Idatha yomkhiqizo

I-T7 RNA Polymerase iyi-polymerase encike ku-DNA ye-RNA evela ku-T7 phage, enomsebenzi oqinile futhi othize ongu-5′→ 3′ RNA polymerase. ifakwe ezansi nomfula isuka kumphromotha.


  • Okwedlule:
  • Olandelayo:

  • Izingxenye

    I-T7 RNA polymerase (50 U/μL)

    10×HH T7 Buffer

    Izimo zokugcina

    Ukuthutha ngaphansi kuka-0°C nokugcinwa ku -25 ~ -15 °C.

    Ukucaciswa

    Igama Lomkhiqizo

    I-T7 RNA Polymerase

    Ukufakwa kwezinhlamvu zomkhiqizo

    Uketshezi olucacile

     

    Umsebenzi wokuchazwa kwencazelo

    Iyunithi eyodwa ichazwa njengenani le-enzyme

    lokho kuzogqugquzela i-NTP ukuthi ikhiqize i-1 nmol PPi in

    Ihora elingu-1 ku-37°C ngaphansi kwesistimu yokusabela evamile.

    Ibhafa yesitoreji

    50 mM Tris-HCl, 100 mM NaCl, 20 mM β-M, 1 mM EDTA, 50% Glycerol ,0.1% (w/v)

    I-Triton® X- 100, pH 7.9 @ ​​25°C.

     

    10×HH T7 ibhafa

    400 mM Tris-HCl (25℃ , pH 8.20) ,60 mM

    I-MgCl2 ,100 mM DTT ,20 mM i-spermidine.

    Isimo sesitoreji

    -25 ~ – 15℃ , gwema ukuncibilika kweqhwa okuphindaphindiwe

     

    Ukusabela kanye Nesimo

    Ukusabela okuvamile

    I-Reagent

    Inani

    Amanzi ane-Nuclease-free H2O noma i-DEPC-Treated

    Kufika ku-20 μL

    10×HH T7 Buffer

    2 ml

    I-ATP/GTP/CTP/UTP (100 mM ngayinye)

    0.4 μL ngayinye (2 mMeach Final)

    I-RNase Inhibitor (40 U/μL) (kuyakhetheka)

    1 μL (40 U)

    I-Pyrophosphatase Inorganic (ngokuzithandela)

    0.5 μL (0.05U)

    I-T7 RNA polymerase (50 U/μL)

    1 μl

    I-Linearized DNA Template

    1 umg

    Engeza izingxenye zokusabela ngendlela engenhla * I-10 × HH T7 Buffer ifaneleka kuphela ama-2mM NTPs, amanye amakhithi okuloba e-High Yield T7 anconyelwa kuma-NTP angu-7.5mM- 10mM.

    Isikhathi Sokufukamela:37 °C amahora ama-2.

    Stop Ukusabela: Engeza u-2μL 0.2 M EDTA (pH8.0@25℃) noma shisisa uye ku-75 °C imizuzu engu-10.

    Ukususwa kwe-DNA: Isifanekiso se-DNA singasuswa nge-2U DNase I (i-RNase-free) kanye ne-incubation imizuzu engu-15 ku-37 °.

     

    Ikhwalithi yokulawula

    Umsebenzi we-Endonuclease: Ukufakwa kokusabela okungu-50μL okuqukethe ubuncane obungu-50U be-T7 RNAPolymerase ne-1 μg λDNA amahora angu-16 ngo-37 ℃ akuphumeleli ukuwohloka okubonakalayo njengoba kunqunyiwe.

    Umsebenzi we-Exonuclease: Ukufakwa kokusabela okungu-50μL okuqukethe ubuncane obungu-50U be-T7 RNA Polymerase eno-1 μg λ -Hind Ⅲ digest DNA amahora angu-16 ngo-37℃ akuphumeleli ukuwohloka okubonakalayo njengoba kunqunyiwe.

    I-Nickase Umsebenzi: Ukufakwa kokusabela okungu-50μL okuqukethe ubuncane obungu-50U be-T7 RNA Polymerase ne-1μg pBR322 DNA amahora angu-16 ku-37°C akuphumeleli ukuwohloka okubonakalayo njengoba kunqunyiwe.

    RNase Umsebenzi: Ukufakwa kokusabela okungu-50μL okuqukethe ubuncane obungu-50U be-T7 RNA Polymerase ne-1.6μg MS2 RNA amahora angu-16 ku-37°C kubangela ukungabikho kokuwohloka okubonakalayo njengoba kunqunyiwe.

    Ukushisa Ukungasebenzi: 75 °C imizuzu engu-10.

     

    Ukuqapha

    • Ukusabela kokulotshiweyo kufanele kwenziwe endaweni engenakho ukungcola kwe-RNase.Ukugqoka amagilavu ​​kuhle.Amathiphu, amashubhu namanzi kufanele kungabi nama-nuclease.

    • Isivuno sokukhiqizwa kwe-RNA singathuthukiswa ngokukhuphula ukugxila kwe-NTP (ukugxilisa ngakunye kungafinyelela ku-10mM), kuyilapho ukugxila kwe-Mg2+ kudinga ukukhushulwa ngokufanelekile.

    • Inhlanganisela ye-RNA synthesis reaction kufanele ilungiswe ekamelweni lokushisa, njengoba i-DNA ingase igibele lapho kukhona 10×HH T7 Buffer ku-4°C.

    •Isivuno sokulotshwa kobude obufanelekile siyehla uma isifanekiso se-DNA sifakwe kulayini ngokungaphelele.•Ingxube yokusabela inganyuswa phezulu noma phansi.• Uma isivuno somkhiqizo wokusabela sehlile, i-20 mM i-DTT entsha ingengezwa ohlelweni lokusabela.

    • Izingcezu ezilotshiwe zingaphansi noma zilingana no-500bp, futhi kuyanconywa ukuthi kunwetshwe isikhathi sokuloba sibe amahora angu-4-8.

    •Izimvula zitholakala kalula ku-10×HH T7.

    Bhala umyalezo wakho lapha futhi usithumelele wona