T7 RNA Polymerase
T7 RNA Polymerase yog ib qho DNA-dependent RNA polymerase los ntawm T7 phage, uas muaj qhov muaj zog thiab tshwj xeeb 5'→ 3' RNA polymerase kev ua haujlwm.T7 RNA Polymerase muaj qhov tshwj xeeb rau T7 cov txheej txheem txhawb nqa thiab yuav ua ke ntau ntau ntawm RNA los ntawm DNA tawg. ntxig downstream los ntawm tus txhawb nqa.
Cheebtsam
T7 RNA polymerase (50 U / μL)
10 × HH T7 Buffer
Cia tej yam kev mob
Kev thauj mus los hauv qab 0 ° C thiab khaws cia ntawm -25 ~ - 15 ° C.
Specification
| Khoom npe | T7 RNA Polymerase |
| Product characterization | Ntshiab kua |
|
Kev ua haujlwm unitdefinition | Ib chav tsev yog txhais raws li tus nqi ntawm enzyme uas yuav catalyze NTP los tsim 1 nmol PPi hauv 1 teev ntawm 37 ° C raws li cov txheej txheem tshuaj tiv thaiv. |
| Cia tsis | 50 mM Tris-HCl, 100 mM NaCl, 20 mM β-M, 1 mM EDTA, 50% Glycerol, 0.1% (w / v) Triton® X-100, pH 7.9 @ 25 ° C. |
|
10 × HH T7 tsis | 400 mM Tris-HCl (25 ℃, pH 8.20), 60 mM MgCl2, 100 mM DTT, 20 mM spermidine. |
| Kev cia khoom | -25 ~ - 15 ℃, tsis txhob rov qab khov-thawing |
Cov tshuaj tiv thaiv thiab mob
Cov tshuaj tiv thaiv kab mob
| Reagent | Tus nqi |
| Nuclease-free H2O lossis DEPC-kho dej | Txog li 20 μL |
| 10 × HH T7 Buffer | 2l miv |
| ATP / GTP / CTP / UTP (100 mM txhua) | 0.4 μL txhua (2 mMeach kawg) |
| RNase Inhibitor (40 U / μL) (yeem) | 1 μL (40 U) |
| Pyrophosphatase Inorganic (yeem) | 0.5 μL (0.05 U) |
| T7 RNA polymerase (50 U / μL) | 1 mL ib |
| Linearized DNA Template | 1m g ib |
Ntxiv cov tshuaj tiv thaiv cov khoom hauv qhov kev txiav txim saum toj no * 10 × HH T7 Buffer tsuas yog tsim rau 2mM NTPs, lwm cov khoom siv High Yield T7 transcription tau pom zoo rau 7.5mM- 10mM NTPs.
Lub sijhawm incubation:37 ° C rau 2 teev.
Stop ntawm Reaction: Ntxiv 2μL 0.2 M EDTA (pH8.0@25 ℃) los yog cua sov rau 75 ° C rau 10min.
Kev tshem tawm DNA: DNA template tuaj yeem raug tshem tawm nrog 2U DNase I (RNase-dawb) thiab incubation rau 15 min ntawm 37 °.
Kev tswj kom zoo
•Endonuclease Activity: Incubation ntawm 50μL cov tshuaj tiv thaiv uas muaj qhov tsawg kawg nkaus ntawm 50U ntawm T7 RNAPolymerase nrog 1 μg λDNA rau 16 teev ntawm 37 ℃ ua rau tsis pom qhov degradation raws li kev txiav txim.
•Kev Ua Haujlwm Exonuclease: Incubation ntawm 50μL cov tshuaj tiv thaiv uas muaj qhov tsawg kawg nkaus ntawm 50U ntawm T7 RNA Polymerase nrog 1 μg λ -Hind Ⅲ zom DNA rau 16 teev ntawm 37 ℃ ua rau tsis pom qhov degradation raws li kev txiav txim.
•Nickase Kev ua si: Incubation ntawm 50μL cov tshuaj tiv thaiv uas muaj qhov tsawg kawg nkaus ntawm 50U ntawm T7 RNA Polymerase nrog 1μg pBR322 DNA rau 16 teev ntawm 37 ° C ua rau tsis pom qhov degradation raws li kev txiav txim.
•RNase Kev ua si: Incubation ntawm 50μL cov tshuaj tiv thaiv uas muaj qhov tsawg kawg nkaus ntawm 50U ntawm T7 RNA Polymerase nrog 1.6μg MS2 RNA rau 16 teev ntawm 37 ° C ua rau tsis pom qhov degradation raws li kev txiav txim.
•Kub InactivationKub: 75 ° C rau 10 min.
Ceev faj
• Cov tshuaj tiv thaiv transcription yuav tsum tau ua nyob rau hauv ib puag ncig tsis muaj RNase paug.Kev hnav hnab looj tes yog pom zoo.Cov lus qhia, raj thiab dej yuav tsum tsis muaj nuclease.
• Kev tsim tawm ntawm RNA tuaj yeem txhim kho los ntawm kev nce qhov concentration ntawm NTP (txhua qhov concentration tuaj yeem ncav cuag 10mM), thaum Mg2+ concentration yuav tsum tau nce kom tsim nyog.
• RNA synthesis cov tshuaj tiv thaiv sib tov yuav tsum tau npaj nyob rau hauv chav tsev kub, vim DNA tej zaum yuav precipitate nyob rau hauv lub xub ntiag ntawm 10 × HH T7 Buffer ntawm 4 ° C.
• Cov txiaj ntsig ntawm cov ntawv sau ntev ntev yuav txo qis yog tias cov qauv DNA tsis ua tiav linearized.• Cov tshuaj tiv thaiv sib xyaw tuaj yeem ua kom nce lossis nqis.• Yog tias cov khoom siv tshuaj tiv thaiv tau txo qis, 20 mM tshiab DTT tuaj yeem muab ntxiv rau hauv cov tshuaj tiv thaiv.
• Cov ntaub ntawv sau tseg tsawg dua lossis sib npaug li 500bp, thiab nws raug nquahu kom ncua lub sijhawm sau ntawv mus rau 4-8 teev.
•Precipitates yooj yim pom nyob rau hauv 10 × HH T7.
