Hi-yield T7 hauv vitro transcription reagent (Thermostable)
Hi-yield T7 hauv vitro transcription reagent cov khoom siv (Thermostable) muaj peev xwm ntawm vitro transcription ntawm qhov kub siab tshaj txog 50 ° C.Cov khoom siv muaj peev xwm ntawm kev sib txuas cov txiaj ntsig siab RNA cov ntaub ntawv siv cov kab tawm ob-stranded DNA uas muaj T7 txhawb nqa ua qauv thiab NTPs ua substrate.Cov khoom siv tsim nyog rau kev koom ua ke ntawm kev hloov kho nucleotides kom tau txais biotin sau npe, dye labeled thiab radiolabeled RNA.Cov khoom siv kuj tuaj yeem siv rau hauv kev sib txuas ntawm co-transcriptionally capping mRNAs nrog cap analogs.Cov khoom siv muaj hloov kho nucleotides N1-Me-pUTP ua lwm txoj.
Txhua tus qauv tshuaj tiv thaiv tawm mus txog 150-200 μg ntawm RNA los ntawm 1μg DNA qauv.Cov tshuaj tiv thaiv loj tuaj yeem ua kom nce mus txog cov txiaj ntsig milligram-theem RNA raws li xav tau.
RNA synthesized los ntawm cov khoom siv yog tsim rau ntau daim ntawv thov nrog rau RNA qauv thiab kev ua haujlwm kev tshawb fawb, ribozyme biochemistry, RNase tiv thaiv kev soj ntsuam thiab hybridization-raws li blots, anti-sense RNA thiab RNAi sim.RNA synthesized los ntawm cov khoom siv kuj yog tsim rau tsim enzymatic zus tau tej cov capped RNA los ntawm cov tshuaj tiv thaiv capping enzyme thiab 2'-O-Methyltransferase thiab raug tailed nrog Poly(A) polymerase los txhim kho stability thiab translational muaj peev xwm ntawm RNA siv rau hauv vitro translation. , transfection, thiab microinjection.
Cheebtsam
| Cheebtsam | Nco ntsoov | Ntim |
| 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-Yield IVT Buffer A | / | 100 μL |
| Enzyme mix (Thermostable) | / | 100 μL |
Cia tej yam kev mob
Kev thauj mus los hauv qab 0 ° C thiab cia ntawm -25 ~ 15 ° C.
Protocal
•DNA template npaj
Linearized plasmid DNA, PCR cov khoom lossis hluavtaws DNA oligonucleotides tuaj yeem siv los ua cov qauv rau hauv vitro transcription nrog cov khoom siv.Cov qauv DNA tuaj yeem yaj hauv TE tsis yog RNase-dawb ddH2O.
•Plasmid cov qauv: Lub linearized plasmid yuav tsum muaj thaj tsam T7 txhawb nqa raws li DNA qauv.Qhov zoo ntawm linearized plasmid cuam tshuam cov txiaj ntsig thiab kev ncaj ncees ntawm RNA.Ua tiav linearized plasmid template ntawm siab tshaj plaws purity yog qhov tseem ceeb rau kev siv cov khoom siv zoo vim tias lub voj voog plasmid tsis ua haujlwm zoo.Qhov siab tshaj plaws transcription yield yog tiav nrog lub siab tshaj plaws purity template.Txhawm rau tsim RNA cov ntawv sau tseg ntawm qhov ntev tau hais tseg, plasmid DNA yuav tsum tau ua tiav linearized nrog kev txwv enzyme uas tsim cov blunt kawg lossis 5'-overhangs.Lub linearized plasmid purified los ntawm kev kuaj sim tuaj yeem tsis muaj kab mob RNase, protein, RNA thiab ntsev.
• PCR templates: PCR cov khoom uas muaj T7 RNA polymerase txhawb nqa nyob rau hauv qhov tseeb kev taw qhia tuaj yeem muab luam tawm.Cov txiaj ntsig zoo dua yuav tau txais nrog cov khoom siv PCR huv si, txawm tias PCR sib xyaw tuaj yeem siv ncaj qha.
• Synthetic DNA oligon uaucleotides:Synthetic DNA oligonucleotides uas yog tag nrho ob-stranded los yog feem ntau ib leeg-stranded nrog ib tug ob-stranded T7 txhawb nqa ib theem zuj zus tuaj yeem luam tawm.
RNA Synthesis raws tu qauv
Hnav hnab looj tes thiab siv cov raj tsis muaj nuclease thiab cov tshuaj reagents kom tsis txhob muaj cov kab mob RNase tau pom zoo.Cov tshuaj tiv thaiv me me yuav tsum tau sib sau ua ke hauv cov raj tsis muaj nuclease microfuge lossis PCR strips.
Conventional RNA Synthesis
1.Thaw cov khoom siv tsim nyog, sib tov thiab mem tes-spin hauv microfuge los sau cov kev daws rau hauv qab ntawm cov hlab.Khaws rau ntawm dej khov.
2.Yog tias koj tab tom npaj yuav khiav ntau qhov kev tawm tsam, nws yog qhov yooj yim los npaj kev sib xyaw ua ke los ntawm kev sib xyaw cov khoom sib npaug ntawm 10 × Hi-Yield IVT Buffer thiab plaub ribonucleotide (NTP) kev daws teeb meem.Siv 10 µl master mix ib zaug.
3. Sib sau cov tshuaj tiv thaiv ntawm chav tsev kub raws li hauv qab no:
| Reagent | Tus nqi |
| Cov dej tsis muaj Nuclease | X μL |
| 10 × Hi-Yield IVT Buffer A | 2l miv |
| ATP / CTP / GTP / UTP * (100 mM txhua) | 2 μL txhua (10 mM txhua qhov kawg) |
| Template DNA | Y μL (1 μg) |
| Enzyme mix (Thermostable) | 2l miv |
| Tag Nrho Cov Lus Teb Volume | 20 mL |
* Txhawm rau txo cov khoom lag luam immunogenicity, UTP tuaj yeem hloov los ntawm N1-Me-pUTP ntawm tib qhov kawg concentration.
4. Sib tov kom huv si, mem tes-spin hauv microfuge.Incubate ntawm 37 ° C rau 2 teev, los yog 50 ° C rau 1 teev yog tias xav tau kev kub ntxhov siab.
5. DNA Digestion: Txhawm rau tshem tawm cov qauv DNA, ntxiv 10U ntawm DNase I rau txhua 20 μL cov tshuaj tiv thaiv, sib tov zoo thiab incubate ntawm 37 ℃ rau 30 feeb.
Capped RNA Synthesis
Zoo sib xws raws tu qauv rau cov pa RNA synthesis, tshwj tsis yog rau cov kauj ruam ntawm assembling cov tshuaj tiv thaiv.Sib sau cov tshuaj tiv thaiv ntawm capped RNA synthesis ntawm chav tsev kub hauv qhov kev txiav txim hauv qab no:
| Reagent | Tus nqi |
| Cov dej tsis muaj Nuclease | X μL |
| 10 × Hi-Yield IVT Buffer A | 2l miv |
| ATP / CTP / GTP / UTP * (100 mM txhua) | 2 μL txhua (10 mM txhua qhov kawg) |
| Cap analog (100 mM) | 1,6l ua |
| Template DNA | Y μL (1 μg) |
| Enzyme mix (Thermostable) | 2l miv |
| Tag Nrho Cov Lus Teb Volume | 20 mL |
*** Yog tias xav tau, Cap1 (3'OH AG) thiab cap1 (3'OMe AG) tuaj yeem siv ascap analogs.Txais tos kom peb cov co-capping reagent Co-capping T7 invitro transcription reagent (pUTP, CAP GAG (3'Ome), pUTP, CAP GAG, N1-Me- pUTP, CAP GAG (3'OMe) thiab N1-Me-pUTP, CAP GAG.
mRNA kev ua kom huv
• Phenol: Chloroform Ntxivkev txiav txim thiab Ethanol Dej nag
Rau kev tshem tawm cov proteins thiab feem ntau ntawm cov nucleotides dawb, phenol: chloroform extraction thiab ethanol nag lossis daus ntawm RNA transcripts yog txoj kev nyiam.
1. Kho cov tshuaj tiv thaiv ntim rau 180 μLby ntxiv 160 μL nuclease-dawb dej.Ntxiv 20 μL ntawm 3M sodium acetate, pH5.2, sib tov kom huv si.
2. Extract nrog sib npaug ntim ntawm 1: 1 phenol / chloroform sib tov, centrifuge ntawm 10000 rpm rau 5 feeb.Sau cov theem aqueous thiab hloov mus rau ib lub raj tshiab.
3. Ua raws li ob qho kev rho tawm nrog qhov sib npaug ntawm chloroform.Sau cov theem aqueous thiab hloov mus rau lub raj tshiab.
4. RNA tau los nag nrog 2 ntim ntawm ethanol.Incubate ntawm -20 ℃ rau yam tsawg kawg 30 feeb, thiab sau lub pallet los ntawm centrifugation rau 15 feeb.Ua tib zoo tshem cov supernatant.
5. Yaug lub pallet nrog 150 μL ~ 200 μL txias 70% ethanol.
6. Cua qhuav lub pallet rau 2 feeb thiab resuspend nyob rau hauv 100 μL ~ 200 μL RNase-dawb dej los yog lwm yam buffers.
• LiCl nag lossis daus
LiCl nag lossis daus ntawm RNA ua haujlwm tau zoo ntawm kev tshem tawm feem ntau ntawm cov NTPs thiab cov enzymes uas tsis koom nrog.
1. Ntxiv qhov sib npaug ntawm LiCl tov (5M), sib tov zoo.
2. Incubate ntawm -20 ℃ rau yam tsawg 30 feeb.Centrifuge ntawm 4 ℃ ntawm 12000 rpm rau 15 feeb rau pallet RNA.Ua tib zoo tshem cov supernatant.
3. Resin lub pallet los ntawm kev ntxiv 200 μL ntawm precooling 70% ethanol.Ua tib zoo tshem tawm cov ethanol.Rov ua cov kauj ruam rau 2 ~ 3 zaug.
4. Cua qhuav lub pallet rau 5 ~ 10 feeb thiab resuspend nyob rau hauv 100 μL ~ 200 μL RNase-dawb dej los yog lwm yam buffers.
• Tig kem purification
Spin kab yuav tshem tawm cov NTPs, cov proteins thiab ntsev uas tsis koom nrog.
• Sib nqus hlaws purintawv tseeb
Magnetic hlaws purification tuaj yeem tshem tawm cov nucleotides tsis sib xws, cov protein thiab ntsev.
Quantification ntawm cov khoom siv tshuaj tiv thaiv
• Quantification los ntawm UV lub teeb absorbance: Cov khoom siv tshuaj tiv thaiv xav tau kev ua kom huv vim tias ib qho tsis muaj nucleotides thiab cov qauv DNA hauv cov tshuaj tiv thaiv sib xyaw yuav cuam tshuam rau kev nyeem ntawv.Kev ntsuas UV spectrophotometric ntawm 260 nm tuaj yeem yooj yim tau txais RNA concentration.Rau ib leeg-stranded RNA, 1 A260 sib raug rau RNA concentration ntawm 40 μg / mL.
• Quantification by xim: Kev ua kom muaj nuj nqis ntawm cov khoom siv tshuaj tiv thaiv tuaj yeem siv nrog Ribogreen dye yam tsis muaj kev ua kom huv vim tias cov nucleotides tsis sib xyaw yuav tsis cuam tshuam rau kev soj ntsuam ntawm cov khoom RNA.
Sau ntawv
• Cov tshuaj tiv thaiv transcription yuav tsum tau ua nyob rau hauv RNase-dawb ib puag ncig.Kev hnav hnab looj tes yog pom zoo.Cov lus qhia, raj thiab dej yuav tsum tsis muaj nuclease.
• Txhua qhov kev pom zoo kawg ntawm NTPs yog 10 mM, thiab koj tuaj yeem hloov qhov kawg concentration ntawm NTPs raws li lub xeev tiag tiag.
• 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 spermidine ntawm 4 ° C.
• Cov txiaj ntsig ntawm cov ntawv sau ntev ntev yuav txo qis yog tias tus qauv DNA tsis ua tiav linearized.
• Cov tshuaj tiv thaiv sib xyaw tuaj yeem ua kom nce lossis nqis.
• Sib tov cov khoom kom txog thaum cov tshuaj insoluble dissolves tag nrho ua ntej siv, yog tias tsis pom muaj cov tshuaj insoluble tom qab remelting.
• Rau cov tshuaj tiv thaiv nrog cov ntawv sau luv luv dua 300 bp, 4-8 h incubation ntawm 37 ℃ yuav tsum muab ntau dua rau koj.
• Cov qauv DNA uas siv rau cov qauv RNA synthesis transcripts yuav tsum muaj T7 txhawb nqa ib ntus ua raws li GGG pib ua ntu zus, vim tias T7 RNA polymerase muaj ntau dua affinity ntawm GTP.
• Cov qauv DNA uas siv rau mRNA synthesis nrog co-transcription system yuav tsum muaj ib tug T7 promoter ib ntus ua raws li AGG pib ua ntu zus.
Kev daws teeb meem
a) Tsawg tawm los ntawm full-length RNA:
Yog hais tias cov tshuaj tiv thaiv transcription generates tag nrho-ntev RNA, tab sis cov txiaj ntsig qis dua li qhov xav tau, nws muaj peev xwm ua kom cov kab mob hauv DNA template inhibiting RNA polymerase, los yog DNA concentration yuav tsawg dhau los yog tsis raug.
Tswv yim: Kev ua kom huv ntxiv ntawm DNA qauv yog pom zoo.
b) RNA cov ntawv sau tseg smear rau denat uauring Gel:
Yog hais tias RNA tshwm sim degraded ntawm denaturing agarose los yog polyacrylamide gel, DNA template los yog cov txheej txheem sim yog kis nrog RNase.
Tswv yim: Yog tias tus qauv plasmid DNA kis tau nrog RNase, ua phenol / chloroform extraction thiab ethanol precipitate.Xyuas kom cov lus qhia thiab cov raj siv thaum lub sij hawm sim yog RNase-dawb.Thov hnav lub tsho lab, daim npog qhov ncauj thiab cov hnab looj tes pov tseg.
c) RNA daim ntawv sau tseg ntawm loj loj tshaj qhov xav tau:
Yog tias cov ntaub ntawv RNA tshwm loj dua li qhov xav tau ntawm denaturing gel, template plasmid DNA yuav tsis ua tiav.Lub xub ntiag ntawm cov txheej txheem thib ob muaj zog tuaj yeem ua rau RNA cov ntawv sau tseg tsis ua tiav tag nrho.
Cov lus qhia: Txheeb xyuas cov qauv rau kev zom zaub mov kom tiav, yog tias tsis pom zoo plasmid raug lees paub, rov qab txwv kev zom zaub mov.Txo cov txheej txheem thib ob ntawm DNA qauv ntawm theem ntawm kev tsim ua ntu zus.
d) RNA cov ntawv sau tseg ntawm me me qhov loj tshaj xav tau:
Yog tias denaturing gel tsom xam pom tias muaj cov kab me me dua li qhov xav tau loj, nws feem ntau yuav yog vim qhov kev txiav tawm ntxov los ntawm polymerase.Qee cov kab ke uas zoo li T7 RNA polymerase txiav tawm cov cim yuav ua rau kev txiav tawm ntxov.
Tswv yim: Incubating transcription reaction at low temperatures, piv txwv li ntawm 30 ° C, yuav ua rau kom qhov feem ntawm tag nrho-ntev transcript, tab sis cov yield yuav raug txo.Rau GC cov qauv nplua nuj, lossis cov qauv nrog cov qauv sib txuas, kev tsim tawm ntawm 42 ° C tuaj yeem txhim kho cov txiaj ntsig ntawm cov ntawv sau ntev.
