
T7 RNA polymerase
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.
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.