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Hi-yield T7 in vitro reagent transcription (kuleylka) HCP0036A Sawirka sifaysan
  • Hi-yield T7 in vitro reagent transcription (kuleylka) HCP0036A

Hi-wax-soo-saarka T7 in vitro reagent transcription (kulka lagu karsado)


Bisadda No.:HCP0036A

Xidhmada:50T

Hi-yield T7 xirmada reagent transcription vitro (Thermostable) ayaa awood u leh in lagu qoro vitro heerkul sare ilaa 50°C.

Sharaxaada Alaabta

Xogta alaabta

Hi-yield T7 xirmada reagent transcription vitro (Thermostable) ayaa awood u leh in lagu qoro vitro heerkul sare ilaa 50°C.Xirmada ayaa awood u leh in ay soo saarto qoraallada RNA ee waxsoosaarka sare leh iyada oo la adeegsanayo DNA-da laba-jibbaaran ee toosan oo ka kooban dhiirrigeliye T7 sida template iyo NTP-yada substrate ahaan.Qalabku wuxuu ku habboon yahay in lagu daro nucleotide la beddelay si loo helo biotin ku calaamadsan, dheeh ku summaysan iyo raadiyaha RNA.Xirmada sidoo kale waxaa lagu dabaqi karaa iyadoo la isku dubariday mRNA-yada koofiyaddaQalabku waxa uu ka kooban yahay nucleotides N1-Me-pUTP beddelka ah.

Falcelinta caadiga ah kasta waxay soo saartaa ilaa 150-200 μg oo RNA ah 1μg qaab DNA ah.Cabbirka falcelinta waa la kordhin karaa si loo soo saaro RNA heerka milligaraam haddii loo baahdo.

RNA laga soo saaray xirmada ayaa ku habboon codsiyo badan oo ay ku jiraan qaab dhismeedka RNA iyo daraasadaha shaqada, ribozyme biochemistry, tijaabooyinka ilaalinta RNase iyo blots-ku-saleysan isku-dhafka, RNA-dareenka-ka-hortagga iyo tijaabada RNAi.RNA laga soo saaray xirmada sidoo kale waxay ku haboon tahay soo saarista enzymatic ee RNA daboolan ee vaccinia caping enzyme iyo 2'-O-Methyltransferase oo lagu dhejiyay Poly(A) polymerase si loo hagaajiyo xasilloonida iyo kartida tarjumaada RNA ee loo isticmaalo tarjumaada vitro , kala-qaadis, iyo irbado yar-yar.


  • Hore:
  • Xiga:

  • Qaybaha

    Qayb

    Xoog-saarid

    Mugga

    ATP

    100 mm

    100 μL

    CTP

    100 mm

    100 μL

    GTP

    100 mm

    100 μL

    UTP

    100 mm

    100 μL

    N1-Me-pUTP

    100 mm

    100 μL

    10 × Hi-Soosaar IVT Buffer A

    /

    100 μL

    Isku darka Enzyme (kuleylka)

    /

    100 μL

     

    Xaaladaha kaydinta

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

     

    Protocal

    • Diyaarinta template DNA

    DNA plasmid Linearized Linearized, PCR ama oligonucleotides DNA synthetic waxaa loo isticmaali karaa qaab-qoraal ah in vitro oo qalabku wato

    Plasmid habyaalada: Plasmiid-ka toosan waa in uu ka kooban yahay gobolka dhiirrigeliyaha T7 sida template DNA.Tayada plasmid-ka toosan waxay saamaysaa wax-soo-saarka iyo daacadnimada RNA.Qaabka plasmid-ka ee nadiifka ah oo dhamaystiran ayaa muhiim u ah isticmaalka guusha leh ee xirmada sababtoo ah plasmid wareegtadu si wax ku ool ah looma joojiyo.Wax-soo-saarka qoraalka ugu sarreeya waxaa lagu gaaraa qaabka ugu nadiifsan ee ugu sarreeya.Si loo soo saaro qoraalka RNA ee dhererka la cayimay, DNA-da plasmid waa in si buuxda loo toosiyaa iyada oo leh ensaym xaddidan oo dhalinaya cidhifyo aan fiicneyn ama 5'-ka-baxsan.Plasmiid-ka toosan ee lagu sifeeyay hababka shaybaadhka waxay ka xoroobi kartaa sumaynta RNase, borotiinka, RNA iyo cusbada.

     Qaababka PCR: Alaabooyinka PCR oo ay ku jiraan dhiirrigeliyaha T7 RNA polymerase ee jihaynta saxda ah waa la qori karaa.Waxsoosaar wanaagsan ayaa lagu heli doonaa alaabada PCR ee la safeeyey, in kasta oo isku darka PCR si toos ah loo isticmaali karo.

    •Synthetic DNA oligonucleotides:DNA oligonucleotides-ka synthetic kuwaas oo ah mid gebi ahaanba laba-jibbaaran ama inta badan hal-xadhig leh oo leh isku xigxiga dhiirrigeliyaha T7 laba-geesoodka ah waa la qori karaa

    Hab-maamuuska RNA Synthesis

    Xidhashada galoofyada iyo isticmaalka tubooyinka iyo reagents-ka ka xorta ah nukliyeerka si looga fogaado faddaraynta RNase ayaa aad loogu talinayaa.Dareen-celinta mugga yar waa in lagu ururiyaa tuubooyinka microfuge-ka-free nuclease-ka ama tuubooyinka fiilooyinka PCR.

    Isku-dhafka RNA ee caadiga ah

    1.Thaw qaybaha xirmada lagama maarmaanka ah, qas iyo garaaca garaaca wadnaha ee microfuge si ay u ururiyaan xal ilaa hoose ee tuubooyinka.Barafka ku hay.

    2.Haddii aad qorsheyneyso inaad sameyso falcelinno badan, way ku habboon tahay inaad diyaariso isku-darka sayidkiisa adoo isku daraya qiyaaso siman oo ah 10 × Hi-Yield IVT Buffer iyo afar ribonucleotide (NTP) xalalka.Isticmaal 10 µl isku darka guud falcelin kasta.

    3. Isku soo ururi falcelinta heerkulka qolka sida soo socota:

    Reagent

    Qadarka

    Biyo aan nukliyeer lahayn

    X μL

    10 × Hi-Soosaar IVT Buffer A

    2 μL

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

    2 μL midkiiba (10 mM final kasta)

    Qaabka DNA-da

    Y μL (1 μg)

    Isku darka Enzyme (kuleylka)

    2 μL

    Wadarta mugga falcelinta

    20 μL

    * Si loo yareeyo difaaca jirka ee sheyga, UTP waxaa lagu bedeli karaa N1-Me-pUTP isla isku uruurinta kama dambaysta ah.

    4. Si fiican isku qas, garaac garaaca garaaca microfuge.Ku duri 37°C 2 saacadood, ama 50°C 1 saac haddii loo baahdo falcelin heerkul sare ah.

    5. Dheefshiidka DNA: Si aad meesha uga saarto DNA-ga template, ku dar 10U of DNAse I si kasta oo 20 μL ah, si fiican isku qas oo ku dhex geli 37℃ 30 daqiiqo.

     

    Qalabaynta RNA la daboolay

    Hab-maamuuska la midka ah isku-dhafka RNA-ga caadiga ah, marka laga reebo tallaabada isu-ururinta falcelinta.Ku soo ururi falcelinta isku-dhafka RNA ee daboolan heerkulka qolka sida soo socota:

    Reagent

    Qadarka

    Biyo aan nukliyeer lahayn

    X μL

    10 × Hi-Soosaar IVT Buffer A

    2 μL

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

    2 μL midkiiba (10 mM final kasta)

    Cap analog (100 mm)

    1.6 μl

    Qaabka DNA-da

    Y μL (1 μg)

    Isku darka Enzyme (kuleylka)

    2 μL

    Wadarta mugga falcelinta

    20 μL

    *** Haddii loo baahdo, Cap1 (3'OH AG) iyo cap1 (3'OMe AG) ayaa loo isticmaali karaa ascap analogs.Waxaa lagu ammaanay isku-dubbarid-ku-qoris isku-duubni T7 reagent reagent (pUTP, CAP GAG (3'OMe) , pUTP, CAP GAG, N1-Me-pUTP, CAP GAG (3'OMe) iyo N1-Me-pUTP, CAP GAG.

     

    nadiifinta mRNA

    Phenol: chloroform Extraficil iyo Ethanol Roobab

    Si loo saaro borotiinnada iyo badi nucleotideyada bilaashka ah, phenol: soo saarista chloroform iyo roobab ethanol ee qoraallada RNA waa habka la door bidayo.

    1. Isku hagaaji mugga falcelinta ilaa 180 μLby adoo ku daraya 160 μL biyo la'aan nukliyeerka.Ku dar 20 μL oo ah 3M sodium acetate, pH5.2, si fiican isku qas.

    2. Soo saar mug siman oo ah 1:1 phenol/ chloroform isku dar ah, sentrifuge at 10000 rpm 5 daqiiqo.Ururi wejiga biyaha oo ku wareeji tuubo cusub.

    3. Waxaa ku xiga laba soosaar oo leh mug siman oo chloroform ah.Ururi wejiga biyaha oo ku wareeji tuubo cusub.

    4. RNA waxa lagu soo degdegay 2 muga ethanol.Ku dubo at -20 ℃ ugu yaraan 30 daqiiqo, ka dibna ku soo ururi palette-centrifugation 15 daqiiqo.Si taxadar leh uga saar waxyaabaha ka sarreeya.

    5. Ku raaci palette-ka 150 μL ~ 200 μL qabow 70% ethanol.

    6. Hawo hawo ku engeji 2 daqiiqo oo dib ugu celi 100 μL ~ 200 μL biyo aan RNase lahayn ama wax kale oo kaydiya.

     

    • Roobka LiCl

    Roobabka LiCl ee RNA waxay wax ku ool u yihiin inay meesha ka saaraan inta badan NTP-yada iyo enzymes-yada aan la isku darin.

    1. Ku dar mugga siman ee xalalka LiCl (5M), si fiican u walaaq.

    2. Ku duri at -20℃ ugu yaraan 30 daqiiqo.Centrifuge at 4 ℃ at 12000 rpm 15 daqiiqo si aad u rogo RNA.Si taxadar leh uga saar waxyaabaha ka sarreeya.

    3. Rekici palette-ka adigoo ku daraya 200 μL oo qaboojin ka hor 70% ethanol.Si taxadar leh uga saar ethanol-ka.Ku celi tillaabooyinka 2-3 jeer.

    4. Hawo hawo ku engeji 5 ~ 10 daqiiqo oo dib ugu celi 100 μL ~ 200 μL biyaha aan RNase-ka lahayn ama meelo kale.

    • Spin safaynta tiirka

    Tiirarka lafdhabarta ayaa ka saari doona NTP-yada aan la isku darin, borotiinnada iyo cusbada.

    • Biraha birta magnetickhayaali

    Sifaynta birta birta ah waxay ka saari kartaa nucleotide, borotiinada iyo cusbada aan la isku darin.

     

    Tiraynta alaabta falcelinta

    • Tirada UV nuugid iftiin: Alaabooyinka falcelinta waxay u baahan yihiin nadiifin sababtoo ah wax kasta oo nucleotide ah oo aan la isku darin iyo DNA-ga qaabaysan ee isku dhafka falcelinta waxay saameyn doontaa akhriska.Cabbiraadda UV spectrophotometry ee 260 nm waxay si fudud u heli kartaa feejignaanta RNA.RNA hal xadhig leh, 1 A260 waxay u dhigantaa fiirsashada RNA ee 40 μg/mL.

     Tirada by dheeh: Qiyaasta alaabta falcelinta waxaa loo isticmaali karaa dheeha Ribogreen iyada oo aan la nadiifin sababtoo ah nucleotides-ka aan la isku darin ma saameyn doonto qiimeynta alaabta RNA.

      

    Xusuusin

    • Dareen-celinta qoraalka waa in lagu sameeyaa deegaan ka xor ah RNase.Xidhashada galoofyada ayaa lagu talinayaa.Talooyinka, tuubooyinka iyo biyuhu waa inay ahaadaan kuwo ka xor ah nukliyeerka.

    Dhammaan uruurinta ugu dambeysa ee NTP-yada ugu fiican waa 10 mM, waxaadna bedeli kartaa uruurinta ugu dambeysa ee NTP-yada iyadoo loo eegayo xaaladda dhabta ah.

    Isku dhafka falcelinta RNA waa in lagu diyaariyaa heerkulka qolka, sababtoo ah DNA-da waxaa laga yaabaa inay soo darto joogitaanka spermidine at 4 ° C.

    • Wax-soosaarka qoraallada dhererka saxda ah ayaa hoos u dhacaya haddii DNA-da qaab-dhismeedka si aan dhammaystirnayn loo sammeeyay.

    Isku darka falcelinta waa la miisaami karaa ama hoos baa loo dhigi karaa.

    • Isku qas bakhaarka ilaa walxaha aan milmi karin ay si buuxda u milmaan ka hor inta aan la isticmaalin, haddii kaydiyaha la ogaado in ay leeyihiin walxo aan milmi karin ka dib marka ay dhalaaliso.

    • Dareen-celinta qoraallada ka gaaban 300 bp, 4-8 saacadood oo ku-tiirsanaanta 37 ℃ waa inay ku siinaysaa wax-soosaar sare.

    Qaabka DNA-da ee loo isticmaalo qoraallada isku-dhafka RNA ee caadiga ah waa inuu ka koobnaadaa taxanaha dhiirrigeliyaha T7 oo ay ku xigto taxanaha bilowga GGG, sababtoo ah T7 RNA polymerase waxay leedahay xidhiidh sare oo GTP ah.

    Qaabka DNA-da ee loo isticmaalo isku-dhafka mRNA ee nidaamka la-qorista waa inuu ka kooban yahay isku xigxiga dhiirrigeliyaha T7 oo ay ku xigto taxanaha bilowga AGG.

    Ciladaynta

    a) Waxsoosaarka hoose ee len buuxagth RNA:

    Haddii falcelinta qoraalku ay dhaliso RNA dhererkeedu dhan yahay, laakiin wax-soo-saarku aad ayuu uga hooseeyaa sidii la filayey, waxaa suurtogal ah in wasakhda ku jirta qaabka DNA-da ay hor istaagto RNA polymerase, ama diiradda DNA-da ayaa laga yaabaa inay aad u hooseyso ama khaldan tahay.

    Soo jeedin: Sifeynta dheeraadka ah ee qaabka DNA-da ayaa lagula talinayaa.

     

    b) RNA qoraal dharbaaxo ilkahaurin Jeel:

    Haddii RNA ay u muuqato mid hoos u dhacday marka la diido agarose ama jel polyacrylamide, template DNA ama habka tijaabada ayaa ku wasakhaysan RNase.

    Soo jeedin: Haddii qaab-dhismeedka DNA-da plasmid-ka uu ku wasakhoobay RNase, samee soo saarista phenol/chloroform iyo soo da'da ethanol.Hubi in talooyinka iyo tuubooyinka la isticmaalo inta lagu jiro tijaabada ay yihiin kuwo ka madax banaan RNase.Fadlan xidho jaakad shaybaadhka, maaskaro iyo galoofyada la tuuri karo.

     

    c) RNA qoraal ah cabbir ka weyn intii la filayey:

    Haddii qoraalka RNA uu u muuqdo mid ka weyn sidii la filayay ee jel-ka-dilid, template plasmid DNA waxa laga yaabaa in aan dhamaystirnayn.Jiritaanka dhismayaal sare oo xooggan ayaa sababi kara qoraalka RNA oo aan gebi ahaanba la diidin.

    Soo jeedin: Hubi qaab-dhismeedka dheefshiidka dhammaystiran, haddii plasmid aan dheefshiidka lahayn la xaqiijiyo, ku celi xakamaynta dheef-shiid kiimikaadka enzyme.Yaree qaababka labaad ee template DNA marxaladda naqshadaynta taxanaha.

     

    d) RNA qoraal ee ka yar cabbirka ka badan la filayo:

    Haddii falanqaynta jel-ka-soo-baxa ay muujiso joogitaanka kooxo yaryar oo ka yar cabbirka la filayo, waxay u badan tahay inay sabab u tahay joojinta degdega ah ee polymerase.Qaar ka mid ah taxanaha u eg T7 RNA polymerase calaamadaha joojinta waxay sababi doonaan joojinta degdega ah.

    Talo soo jeedin: Ku kicinta falcelinta qoraalka ee heerkul hoose, tusaale ahaan 30°C, waxay kordhin kartaa saamiga qoraalka dhererka buuxa, si kastaba ha ahaatee wax-soosaarka waa la dhimi doonaa.Qaababka hodanka ah ee GC, ama qaab-dhismeedka leh qaab-dhismeedka sare, ku-wareejinta 42°C waxay wanaajin kartaa dhalidda qoraalka dhererka buuxa.

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