I-T7 RNA Polymerase
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.
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.