I-Hi-yield T7 in vitro transcription reagent (Thermostable)
Ikhithi ye-Hi-yield T7 in vitro transcription reagent (Thermostable) iyakwazi ukulotshwa nge-in vitro emazingeni okushisa aphakeme afika ku-50°C.Ikhithi iyakwazi ukuhlanganisa imibhalo ye-RNA enesivuno esikhulu isebenzisa i-DNA enemicu ephindwe kabili equkethe iphromotha ye-T7 njengesifanekiso kanye nama-NTP njenge-substrate.Ikhithi ifanele ukufakwa kwama-nucleotide aguquliwe ukuze kutholwe i-biotin enelebuli, udayi obhalwe ukuthi i-RNA ene-radiolabeled.Ikhithi futhi ingasetshenziswa ekuhlanganiseni kwe-co-transcriptionally capping mRNAs nama-cap analogs.Ikhithi iqukethe ama-nucleotide aguquliwe i-N1-Me-pUTP njengenye indlela.
Ukusabela ngakunye okujwayelekile kukhiqiza kufika ku-150-200 μg we-RNA kusuka ku-1μg yesifanekiso se-DNA.Usayizi wokusabela ungalinganiswa ukuze ukhiqize i-RNA yezinga le-milligram njengoba kudingeka.
I-RNA ehlanganiswe kusukela kukhithi ifaneleka ezinhlelweni eziningi ezihlanganisa ukwakheka kwe-RNA kanye nezifundo zokusebenza, i-ribozyme biochemistry, izivivinyo zokuvikela i-RNase nama-blots asekelwe ekuhlanganiseni, i-anti-sense RNA kanye nokuhlolwa kwe-RNAi.I-RNA ehlanganiswe kusuka kukhithi futhi ilungele ukukhiqiza ukukhiqizwa kwe-enzymatic ye-RNA ehlanganisiwe nge-vaccinia capping enzyme kanye ne-2'-O-Methyltransferase futhi ihlanganiswe ne-Poly(A) polymerase ukuthuthukisa ukuzinza nekhono lokuhumusha le-RNA esetshenziselwa ukuhumusha in vitro. , transfection, kanye microinjection.
Izingxenye
Isakhi | Ukugxila | Ivolumu |
I-ATP | 100 mM | 100 μL |
I-CTP | 100 mM | 100 μL |
I-GTP | 100 mM | 100 μL |
I-UTP | 100 mM | 100 μL |
I-N1-Me-pUTP | 100 mM | 100 μL |
10 × I-Hi-Yield IVT Buffer A | / | 100 μL |
Ingxube ye-enzyme (Thermostable) | / | 100 μL |
Izimo zokugcina
Ukuhamba ngaphansi kuka-0°C nokugcinwa ku- -25~- 15°C.
I-Protocal
•Ukulungiswa kwesifanekiso se-DNA
I-plasmid DNA ewumugqa, imikhiqizo ye-PCR noma i-DNA oligonucleotide yokwenziwa ingasetshenziswa njengezifanekiso zokulotshwa kwe-in vitro ngekhithi.Isifanekiso se-DNA singancibilika ku-TE buffer noma ku-ddH2O yamahhala ye-RNase.
•I-Plasmid izifanekiso: I-plasmid yomugqa kufanele iqukathe isifunda sephromotha ye-T7 njengesifanekiso se-DNA.Ikhwalithi ye-plasmid yomugqa ithinta isivuno nobuqotho be-RNA.Isifanekiso se-plasmid esenziwe umugqa ngokuphelele sokuhlanzeka okuphezulu sibalulekile ekusetshenzisweni ngempumelelo kwekhithi ngoba i-plasmid eyindilinga ayinqanyulwa ngempumelelo.Isivuno esiphezulu sokulotshwa sitholwa ngesifanekiso sokuhlanzeka esiphezulu.Ukuze kukhiqizwe umbhalo we-RNA wobude obuchaziwe, i-plasmid DNA kufanele ihambisane ngokuphelele ne-enzyme evimbelayo ekhiqiza iziphetho ezibuthuntu noma ama-overhang angu-5'.I-plasmid yomugqa ehlanjululwe ngezindlela zaselabhorethri ingakhululeka ekungcoliseni i-RNase, amaprotheni, i-RNA nosawoti.
• Izifanekiso ze-PCR: Imikhiqizo ye-PCR equkethe iphromotha ye-T7 RNA polymerase isendaweni efanele ingabhalwa.Isivuno esingcono sizotholakala ngemikhiqizo ye-PCR ehlanziwe, nakuba izingxube ze-PCR zingasetshenziswa ngokuqondile.
•Ezokwenziwa I-DNA i-oligonama-ucleotides:I-synthetic DNA oligonucleotides enemicu ephindwe kabili noma iningi umucu owodwa nokulandelana komgqugquzeli we-T7 onemicu emibili ingabhalwa.
I-RNA Synthesis Protocols
Ukugqoka amagilavu nokusebenzisa amashubhu nama-reagents angenayo i-nuclease ukugwema ukungcoliswa kwe-RNase kunconywa kakhulu.Ukusabela kwevolumu encane kufanele kuhlanganiswe kumashubhu e-nuclease-free microfuge noma amashubhu e-PCR strip.
I-RNA Synthesis evamile
1.Ncibilikisa izakhi zekhithi ezidingekayo, xuba futhi ushaye-spin ku-microfuge ukuze uqoqe izixazululo phansi kwamashubhu.Qhubeka eqhweni.
2.Uma uhlela ukusebenzisa ukusabela okuningi, kulula ukulungisa imiksi eyinhloko ngokuhlanganisa amavolumu alinganayo e-10× Hi-Yield IVT Buffer kanye nezixazululo ezine ze-ribonucleotide (NTP).Sebenzisa i-10 µl master mix ngokusabela ngakunye.
3. Hlanganisa ukusabela ekamelweni lokushisa ngendlela elandelayo:
I-Reagent | Inani |
Amanzi angenayo i-nuclease | X μL |
10 × I-Hi-Yield IVT Buffer A | 2 ml |
I-ATP/CTP/GTP/UTP *(100 mM ngayinye) | 2 μL ngayinye(10 mM okokugcina ngakunye) |
I-DNA yesifanekiso | Y μL (1 μg) |
Ingxube ye-enzyme (Thermostable) | 2 ml |
Ivolumu Yokusabela Ephelele | 20 μL |
* Ukunciphisa ukuzivikela komkhiqizo, i-UTP ingathathelwa indawo yi-N1-Me-pUTP ekugxiliseni okufanayo kokugcina.
4. Hlanganisa kahle, i-pulse-spin ku-microfuge.Fukamela ku-37°C amahora angu-2, noma u-50 °C ihora eli-1 uma ukusabela okuphezulu kwezinga lokushisa kuyadingeka.
5. Ukugaya Kwe-DNA: Ukuze ukhiphe isifanekiso se-DNA, engeza i-10U ye-DNase I ekuphenduleni ngakunye okungu-20 μL, hlanganisa kahle futhi ufukamele ku-37℃ imizuzu engu-30.
I-Capped RNA Synthesis
Iphrothokholi efanayo ekuhlanganiseni kwe-RNA evamile, ngaphandle kwesinyathelo sokuhlanganisa ukusabela.Hlanganisa ukusabela kwe-capped RNA synthesis ekamelweni lokushisa ngohlelo olulandelayo:
I-Reagent | Inani |
Amanzi angenayo i-nuclease | X μL |
10 × I-Hi-Yield IVT Buffer A | 2 ml |
I-ATP/CTP/GTP/UTP *(100 mM ngayinye) | 2 μL ngayinye (10 mM Final ngayinye) |
I-analog cap (100 mM) | 1.6 μL |
I-DNA yesifanekiso | Y μL (1 μg) |
Ingxube ye-enzyme (Thermostable) | 2 ml |
Ivolumu Yokusabela Ephelele | 20 μL |
*** Uma kudingeka, i-Cap1(3'OH AG) kanye ne-cap1(3'OMe AG) zingasetshenziswa ama-ascap analog.Sincome i-co-transcription reagent yethu i-Co-capping T7 invitro transcription reagent (pUTP, CAP GAG (3'OMe) , pUTP, CAP GAG, N1-Me- pUTP, CAP GAG (3'OMe) kanye ne-N1-Me-pUTP, CAP I-GAG.
ukuhlanzwa kwe-mRNA
• I-Phenol: I-Chloroform Extraisenzo kanye ne-Ethanol Imvula
Ukuze kukhishwe amaprotheni kanye neningi lama-nucleotide amahhala, i-phenol: i-chloroform extraction kanye nemvula ye-ethanol yemibhalo ye-RNA iyindlela ekhethwayo.
1.Lungisa ivolumu yokusabela ibe ngu-180 μL ngokungeza u-160 μL amanzi angenayo i-nuclease.Engeza u-20 μL we-3M sodium acetate, pH5.2, hlanganisa kahle.
2. Khipha ngevolumu elinganayo yengxube ye-phenol/chloroform engu-1:1, i-centrifuge ku-10000 rpm imizuzu emi-5.Qoqa isigaba se-aqueous bese udlulisela ku-tube entsha.
3. Kulandelwa izingcaphuno ezimbili ezinomthamo olinganayo we-chloroform.Qoqa isigaba se-aqueous bese udlulisela ku-tube entsha.
4. I-RNA yancipha ngemiqulu emi-2 ye-ethanol.Fukamela ku -20 ℃ okungenani imizuzu engama-30, bese uqoqa i-pallet nge-centrifugation imizuzu engu-15.Susa ngokucophelela amandla angaphezu kwavamile.
5. Hlanza i-pallet nge-150 μL~200 μL ebandayo engu-70% ye-ethanol.
6. Yomisa i-pallet emoyeni imizuzu emi-2 bese uphinda uyimise emanzini angu-100 μL~200 μL RNase-free noma ezinye izibhafa.
• Imvula ye-LiCl
Imvula ye-LiCl ye-RNA iyasebenza ekususeni iningi lama-NTP nama-enzyme angahlanganisiwe.
1. Engeza umthamo olinganayo wesixazululo se-LiCl (5M), hlanganisa kahle.
2. Fudumeza ku -20℃ okungenani imizuzu engama-30.I-Centrifuge ku-4 ℃ ku-12000 rpm imizuzu engu-15 ukuya ku-pallet RNA.Susa ngokucophelela amandla angaphezu kwavamile.
3. Geza i-pallet ngokungeza u-200 μL we-ethanol engu-70%.Khipha ngokucophelela i-ethanol.Phinda izinyathelo izikhathi ezingu-2-3.
4. Yomisa i-pallet emoyeni imizuzu engu-5 ~ 10 bese uphinda uyimise emanzini angu-100 μL~200 μL RNase-free noma ezinye izibhafa.
• Spin ukuhlanzwa kwekholomu
Spin amakholomu azosusa ama-NTP angahlanganisiwe, amaprotheni nosawoti.
• I-puri yobuhlalu obuzibutheinganekwane
Ukuhlanzwa kobuhlalu obuzibuthe kungasusa ama-nucleotide angahlanganisiwe, amaprotheni nosawoti.
Ukulinganisa kwemikhiqizo yokusabela
• Ukulinganisa nge-UV ukumunca ukukhanya: Imikhiqizo yokusabela idinga ukuhlanzwa ngoba noma imaphi ama-nucleotide angahlanganisiwe kanye ne-DNA yesifanekiso kuzingxube zokusabela kuzothinta ukufundwa.Ukulinganisa i-spectrophotometry ye-UV ku-260 nm kungathola kalula ukugxila kwe-RNA.Ku-RNA enomucu owodwa, i-1 A260 ihambisana nokugxila kwe-RNA okungu-40 μg/mL.
• Quantification by udayi: Ukulinganisa kwemikhiqizo yokusabela kungasetshenziswa ngodayi we-Ribogreen ngaphandle kokuhlanzwa ngoba ama-nucleotide angahlanganisiwe ngeke athinte ukuhlolwa kwemikhiqizo ye-RNA.
Amanothi
• Ukusabela kokulotshiweyo kufanele kwenziwe endaweni engenayo i-RNase.Ukugqoka amagilavu kuhle.Amathiphu, amashubhu namanzi kufanele kungabi nama-nuclease.
• Konke ukugxiliswa kokugcina okuphelele kwama-NTP ngu-10 mM, futhi ungashintsha ukugxiliswa kokugcina kwama-NTP ngokuya ngesimo sangempela.
• Inhlanganisela ye-RNA synthesis reaction kufanele ilungiswe ekamelweni lokushisa, njengoba i-DNA ingase igibele lapho kukhona i-spermidine ku-4°C.
• Isivuno sokulotshwa kobude obufanelekile siyehla uma isifanekiso se-DNA sifakwe kulayini ngokungaphelele.
• Ingxube yokusabela ingakhuliswa phezulu noma phansi.
• Hlanganisa isigcinalwazi kuze kube yilapho izinto ezingancibiliki zincibilika ngokuphelele ngaphambi kokusetshenziswa, uma isigcinalwazi sitholakala sinezinto ezingancibiliki ngemva kokuncibilika kabusha.
• Ngokusabela okulotshiweyo okufushane kuno-300 bp, ukufukamela kwamahora angu-4-8 ku-37 ℃ kufanele kukunike isivuno esiphezulu.
• Isifanekiso se-DNA esisetshenziselwa imibhalo evamile ye-RNA synthesis kufanele siqukathe ukulandelana kwephromotha ye-T7 elandelwa ukulandelana kokuqala kwe-GGG, ngoba i-T7 RNA polymerase inokuhambisana okuphezulu kwe-GTP.
• Isifanekiso se-DNA esisetshenziselwa ukuhlanganiswa kwe-mRNA ne-co-transcription system kufanele siqukathe ukulandelana kwephromotha ye-T7 elandelwa ukulandelana kokuqala kwe-AGG.
Ukuxazulula inkinga
a) Isivuno esiphansi se-full-length RNA:
Uma ukusabela okulotshiweyo kukhiqiza i-RNA yobude obugcwele, kodwa isivuno siphansi kakhulu kunokulindelekile, kungenzeka ukuthi ukungcola kusifanekiso se-DNA kuvimbela i-RNA polymerase, noma ukugxiliswa kwe-DNA kungase kube phansi kakhulu noma kungalungile.
Ukusikisela: Kunconywa ukuhlanzwa okwengeziwe kwesifanekiso se-DNA.
b) I-RNA okulotshiweyo ukugcoba denatukuchama Ijeli:
Uma i-RNA ibonakala yehlisiwe kujeli ye-denaturing agarose noma ye-polyacrylamide, isifanekiso se-DNA noma inqubo yokuhlola ingcoliswe yi-RNase.
Ukusikisela: Uma isifanekiso se-plasmid DNA singcoliswe yi-RNase, yenza i-phenol/chloroform extract and ethanol precipitate.Qinisekisa ukuthi amathiphu namashubhu asetshenziswa ngesikhathi sokuhlolwa awanayo i-RNAse.Sicela ugqoke ijazi laselabhu, imaski namagilavu alahlwayo.
c) RNA okulotshiweyo kwe usayizi omkhulu kunokulindelekile:
Uma umbhalo we-RNA ubonakala umkhulu kunokulindelekile kujeli ye-denaturing, i-plasmid DNA yesifanekiso ingase ingagayeki ngokuphelele.Ukuba khona kwezakhiwo zesibili eziqinile kungabangela okulotshiweyo kwe-RNA ukuthi kungabi yi-denatured ngokuphelele.
Ukusikisela: Hlola isifanekiso ukuze uthole ukugayeka kokudla okuphelele, uma i-plasmid engagayekile iqinisekiswa, phinda ukugayeka kokudla kwe-enzyme.Yehlisa izakhiwo zesibili zesifanekiso se-DNA esigabeni sokuklama ngokulandelana.
d) I-RNA okulotshiweyo kwe ezincane usayizi kuna okulindelekile:
Uma ukuhlaziywa kwejeli ye-denaturing kubonisa ukuba khona kwamabhendi amancane kunosayizi olindelekile, cishe kungenxa yokunqanyulwa ngaphambi kwesikhathi yi-polymerase.Okunye ukulandelana okufana nezimpawu zokunqanyulwa kwe-T7 RNA polymerase kuzodala ukunqanyulwa ngaphambi kwesikhathi.
Isiphakamiso: Ukufukamela ukusabela kokulotshiweyo emazingeni okushisa aphansi, isibonelo ku-30°C, kungase kwenyuse ingxenye yombhalo wobude obugcwele, kodwa isivuno sizoncishiswa.Ezifanekisweni ezinothe ze-GC, noma izifanekiso ezinezakhiwo zesibili, ukufukamela kokuthi 42°C kungase kuthuthukise isivuno sombhalo wobude obugcwele.