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T7 RNA Polymerase HCP1011A Sawirka Muuqaalka ah
  • T7 RNA Polymerase HCP1011A

T7 RNA polymerase


Bisadda No.:HCP1011A

Xidhmada: 100μL/1ml/10ml/100ml

T7 RNA Polymerase waa RNA polymerase ku tiirsan DNA oo ka yimid T7 phage

Sharaxaada Alaabta

Xogta alaabta

T7 RNA Polymerase waa DNA-ku-tiirsan RNA polymerase ka T7 phage, kaas oo leh firfircooni xoog leh oo gaar ah 5′→ 3′ RNA polymerase. la geliyo dhinaca hoose ee ka soo jeediyaha.


  • Hore:
  • Xiga:

  • Qaybaha

    T7 RNA polymerase (50 U/μL)

    10×HH T7 Buffer

    Xaaladaha kaydinta

    Gaadiidka ka hooseeya 0°C iyo kaydinta at -25 ~ -15°C.

    Tilmaamid

    Magaca Alaabta

    T7 RNA polymerase

    Tilmaamaha alaabta

    dareere cad

     

    Qeexid hawleed midaysan

    Hal unug ayaa lagu qeexaa cadadka insaymiska

    taas oo kicin doonta NTP si ay u soo saarto 1 nmol PPi gudaha

    1 saac at 37°C hoos yimaada nidaamka falcelinta caadiga ah.

    Kaydinta kaydinta

    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 kaydiye

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

    MgCl2,100 mM DTT,20mM shahwada.

    Xaaladda kaydinta

    -25 ~ - 15 ℃, iska ilaali qaboojinta soo noqnoqda

     

    Falcelinta iyo Xaaladda

    Falcelinta caadiga ah

    Reagent

    Qadarka

    H2O-free nucleus ama biyo-daaweeyay DEPC

    Ilaa 20 μl

    10×HH T7 Buffer

    2 μL

    ATP/GTP/CTP/UTP (100 mM midkiiba)

    0.4 μL midkiiba (2 mMeach Final)

    RNase Inhibitor (40 U/μL) (ikhtiyaar)

    1 μL (40 U)

    Pyrophosphatase Inorganic (ikhtiyaar ah)

    0.5 μL (0.05U)

    T7 RNA polymerase (50 U/μL)

    1 μL

    Qaabka DNA oo toosan

    1 μg

    Ku dar qaybaha falcelinta ee habka kore * 10 × HH T7 Buffer wuxuu ku habboon yahay 2mM NTPs, xirmooyinka kale ee Wax-soosaarka Sare ee T7 ayaa lagu talinayaa 7.5mM-10mM NTPs.

    Waqtiga soo galitaanka:37 °C ilaa 2 saacadood.

    Stop falcelinta: Ku dar 2μL 0.2 M EDTA (pH8.0@25℃) ama kuleyl 75 °C 10min.

    Saaridda DNA: Qaabka DNA-da waxaa lagu saari karaa 2U DNAse I (RNase-free) iyo ku-kuleejinta 15 min at 37 °.

     

    Xakamaynta tayada

    Dhaqdhaqaaqa Endonuclease: Kordhinta falcelinta 50μL oo ka kooban ugu yaraan 50U ee T7 RNAPolymerase leh 1 μg λDNA 16 saacadood at 37 ℃ natiijadu ma keenayso hoos u dhac la ogaan karo sida la go'aamiyey.

    Waxqabadka Exonuclease: Kordhinta falcelinta 50μL oo ka kooban ugu yaraan 50U ee T7 RNA Polymerase leh 1 μg λ -Hind Ⅲ dheefshiidka DNA 16 saacadood 37 ℃ natiijadu ma keenayso hoos u dhac la ogaan karo sida la go'aamiyay.

    Nickase Hawsha: Kordhinta falcelinta 50μL oo ka kooban ugu yaraan 50U ee T7 RNA Polymerase leh 1μg pBR322 DNA 16 saacadood at 37°C natiijadu ma keenayso hoos u dhac la ogaan karo sida la go'aamiyay.

    RNase Hawsha: Kordhinta falcelinta 50μL oo ka kooban ugu yaraan 50U ee T7 RNA Polymerase leh 1.6μg MS2 RNA 16 saacadood at 37°C natiijadu ma keenayso hoos u dhac la ogaan karo sida la go'aamiyay.

    Dhaqdhaqaaq la'aanta kulaylka: 75 °C 10 daqiiqo.

     

    Ka taxaddar

    • Dareen-celinta qoraal-celinta waa in lagu sameeyaa deegaan ka madaxbannaan sumowga RNase.Xidhashada galoofyada ayaa lagu talinayaa.Talooyinka, tuubooyinka iyo biyuhu waa inay ka xoroobaan nukliyeerka.

    • Wax-soo-saarka RNA waxaa lagu wanaajin karaa iyadoo la kordhinayo xoogga saarista NTP (fiiritaan kasta wuxuu gaari karaa 10mM), halka xoojinta Mg2+ loo baahan yahay in si habboon loo kordhiyo.

    Isku dhafka falcelinta RNA waa in lagu diyaariyaa heerkulka qolka, maadaama DNA-da laga yaabo inay soo darto joogitaanka 10 × HH T7 Buffer at 4°C.

    • Wax-soo-saarka qoraallada saxda ah ee dhererka saxda ah ayaa hoos u dhacaya haddii DNA-da qaab-dhismeedku uu yahay mid aan dhammaystirnayn oo toosan.Isku darka falcelinta waa la kordhin karaa ama hoos baa loo dhigi karaa.Haddii wax-soo-saarka fal-celinta uu yaraado, 20 mm DTT cusub ayaa lagu dari karaa habka falcelinta.

    • Jajabyada la qoray waxay ka yar yihiin ama la mid yihiin 500bp, waxaana lagu talinayaa in la kordhiyo wakhtiga qoraalka ilaa 4-8 saacadood.

    • Da'ay waa sahlan tahay in laga helo 10×HH T7.

    Halkan ku qor fariintaada oo noo soo dir