Sterilization ntawm cov lus qhia pipette thiab EP raj, thiab lwm yam.
1. Npaj 0.1% (ib txhiab) DEPC (cov khoom muaj kuab lom heev) nrog dej deionized, ua tib zoo siv hauv lub hood fume, thiab khaws cia ntawm 4 ° C deb ntawm lub teeb;
DEPC dej yog dej ntshiab uas kho nrog DEPC thiab sterilized los ntawm qhov kub thiab txias.Kev ntsuam xyuas kom tsis pub muaj RNase, DNase thiab proteinase.
2. Muab lub taub hau thiab EP raj tso rau hauv 0.1% DEPC, thiab xyuas kom meej tias lub raj xa dej thiab EP raj muaj 0.1% DEP.
3. Tiv thaiv los ntawm lub teeb, cia sawv ntsug, hmo ntuj (12-24 teev)
4. Lub thawv uas muaj lub hau thiab EP raj tsis tas yuav tsum tau tsau rau hauv DEPC.Tom qab tshem tawm cov dej DEPC hauv lub taub lossis EP raj, ntim nws thiab qhwv nws.
5. 121 ° C, 30 min
6. 180 degrees Celsius, qhuav rau ob peb teev (tsawg kawg 3 teev)
Nco tseg: a.Hnav hnab looj tes thiab lub qhov ncauj qhov ntswg thaum tuav DEPC!b, los yog tsis muaj DEPC sterilization, 130 ℃, 90min autoclave (ntau lub chaw soj nstuam kub sterilization ob zaug)
RNA extraction xav txog
Ob qhov tshwm sim loj ntawm cov ntaub so ntswg RNA cais tsis ua haujlwm
RNA degradation thiab residue ntawm impurities hauv cov ntaub so ntswg,hais txog kev degradation, cia peb saib ua ntej vim li cas RNA rho tawm los ntawm kab lis kev cai tsis yooj yim degraded.Tam sim no RNA extraction reagents tag nrho muaj cov khoom uas inhibit RNase sai.Ntxiv cov lysate rau cov kab lis kev cai, thiab tsuas yog sib tov nws, tag nrho cov hlwb tuaj yeem sib xyaw nrog cov lysate, thiab cov hlwb tau lysed tag.Tom qab cov hlwb raug lysed, cov khoom xyaw nquag hauv lysate tam sim ntawd inhibit intracellular RNase, yog li RNA tseem nyob twj ywm.Qhov ntawd yog hais tias, vim hais tias cov kab lis kev cai tau yooj yim thiab muaj kev sib cuag nrog cov lysate, lawv RNA tsis yooj yim degraded;Ntawm qhov tod tes, RNA nyob rau hauv cov ntaub so ntswg yog yooj yim degraded vim hais tias cov hlwb nyob rau hauv cov ntaub so ntswg tsis yooj yim mus cuag cov lysate sai.vim muaj kev sib cuag txaus.Yog li ntawd,Piv txwv tias muaj ib txoj hauv kev los hloov cov ntaub so ntswg mus rau hauv ib lub cell thaum inhibiting RNA kev ua, qhov teeb meem ntawm degradation yuav daws tau tag nrho.
Cov kua nitrogen milling yog txoj kev zoo tshaj plaws.Txawm li cas los xij, txoj kev ua kua nitrogen milling yog teeb meem heev, tshwj xeeb tshaj yog thaum muaj pes tsawg tus qauv loj.Qhov no ua rau qhov zoo tshaj plaws tom ntej: homogenizer.Covhomogenizertxoj kev tsis xav txog cov lus nug ntawm yuav ua li cas RNase kev ua haujlwm yog inhibited ua ntej cov hlwb raug tiv tauj nrog lysate, tab sis theej thov Vajtswv tias tus nqi ntawm cov ntaub so ntswg cuam tshuam sai dua li tus nqi ntawm intracellular RNase degrades RN.
Cov nyhuv ntawm hluav taws xob homogenizer zoo dua,thiab cov nyhuv ntawm iav homogenizer tsis zoo, tab sis feem ntau, homogenizer txoj kev tsis tuaj yeem tiv thaiv qhov tshwm sim degradation.Yog li ntawd, yog hais tias lub extraction yog degraded, tus thawj hluav taws xob homogenizer yuav tsum tau siv rau kev sib tsoo nrog kua nitrogen;thawj iav homogenizer yuav tsum tau hloov mus rau ib qho hluav taws xob homogenizer los yog ncaj qha milled nrog kua nitrogen.Qhov teeb meem yog yuav luag 100% ua tau.daws.
Cov teeb meem impurity residue cuam tshuam rau cov kev sim tom ntej muaj ntau yam ua rau ntau dua li degradation, thiab cov kev daws teeb meem sib txawv.Hauv kev xaus,Yog hais tias muaj degradation los yog residual impurities nyob rau hauv cov ntaub so ntswg, txoj kev extraction / reagent rau tej yam kev sim cov khoom yuav tsum optimized.Koj tsis tas yuav siv koj cov qauv zoo rau kev ua kom zoo: koj tuaj yeem yuav qee cov tsiaj me xws li ntses / nqaij qaib los ntawm kev ua lag luam, coj ib feem ntawm cov khoom siv rau RNA extraction, thiab lwm qhov rau kev rho tawm cov protein - zom nrog lub qhov ncauj, plab thiab hnyuv Extract.
Lub hom phiaj RNA ntawm RNA rho tawm yog siv rau kev sib txawv ntawm kev soj ntsuam, thiab nws cov kev xav tau zoo sib txawv
cDNA tsev qiv ntawv kev tsim kho yuav tsum muaj RNA kev ncaj ncees yam tsis muaj residues ntawm cov tshuaj tiv thaiv enzyme inhibitors;Sab qaum teb yuav tsum muaj kev ncaj ncees RNA siab dua thiab qis dua rau cov tshuaj tiv thaiv enzyme inhibitors residues;RT-PCR tsis xav tau RNA siab dhau lawm,tab sis inhibits cov tshuaj tiv thaiv enzyme.Residue yuav tsum nruj.Lub tswv yim txiav txim qhov tso zis;txhua lub sij hawm lub hom phiaj yog kom tau lub siab tshaj plaws purity RNA, nws yuav raug nqi rau tib neeg thiab nyiaj txiag.
Kev sau / khaws cov qauv
Cov yam ntxwv cuam tshuam rau Degradation Tom qab tus qauv tawm hauv lub cev nyob / lossis qhov chaw loj hlob qub, cov enzymes endogenous hauv cov qauv yuav pib degrade RNA,thiab tus nqi degradation muaj feem xyuam rau cov ntsiab lus ntawm endogenous enzymes thiab kub.Kev lig kev cai, tsuas muaj ob txoj hauv kev los ua kom tag nrho inhibit endogenous enzyme kev ua: ntxiv lysate tam sim ntawd thiab homogenize kom huv si thiab sai;txiav rau hauv me me pieces thiab tam sim ntawd khov nyob rau hauv kua nitrogen.Ob txoj hauv kev xav tau kev ua haujlwm ceev.Cov yav tas yog haum rau tag nrho cov qauv, thaum lub qub tsuas yog haum rau cov ntaub so ntswg uas tsis muaj cov ntsiab lus ntawm cov hlwb thiab endogenous enzymes thiab yooj yim rau homogenize.Tshwj xeeb, cov ntaub so ntswg, daim siab, thymus, pancreas, spleen, hlwb, rog, nqaij nqaij, thiab lwm yam yog qhov zoo tshaj plaws khov nrog kua nitrogen ua ntej ua haujlwm.
Fragmentation thiab homogenization ntawm cov qauv
Cov yam ntxwv cuam tshuam rau kev degradation thiab tawm los qauv fragmentation yogkom meej homogenization, uas yog ua kom tiav thiab ua tiav tso tawm ntawm RNA.Cells tuaj yeem ncaj qha homogenized yam tsis tau tawg.Cov ntaub so ntswg tuaj yeem ua homogenized tsuas yog tom qab tawg.Cov poov xab thiab cov kab mob yuav tsum tau tawg nrog cov enzymes sib xws ua ntej lawv tuaj yeem ua homogenized.Cov ntaub so ntswg nrog qis endogenous enzyme cov ntsiab lus thiab yooj yim homogenization yuav crushed thiab homogenized nyob rau hauv ib lub sij hawm nyob rau hauv lub lysate los ntawm ib tug homogenizer;cov ntaub so ntswg, daim siab, thymus, pancreas, spleen, hlwb, rog, nqaij nqaij thiab lwm yam qauv, Lawv yog cov siab nyob rau hauv endogenous enzymes los yog tsis yooj yim homogenized,yog li cov ntaub so ntswg cuam tshuam thiab homogenization yuav tsum tau ua nyias.Txoj kev txhim khu kev qha tshaj plaws thiab ua tau zoo tshaj plaws ntawm kev tawg yog milling nrog kua nitrogen, thiab txoj kev ntseeg siab tshaj plaws ntawm homogenization yog kev siv hluav taws xob homogenizer.Ib daim ntawv tshwj xeeb txog kev milling nrog kua nitrogen: cov qauv yuav tsum tsis txhob thawed thaum lub sij hawm tag nrho cov txheej txheem milling, vim endogenous enzymes muaj feem yuav ua hauj lwm thaum khov.
Kev xaiv ntawm lysate
Kev cuam tshuam rau kev yooj yim ntawm kev ua haujlwm thiab cov yam ntxwv ntawm cov khoom seem endogenous impurities Cov kev siv lysis feem ntau tuaj yeem cuam tshuam cov haujlwm ntawm RNase.Yog li ntawd, lub ntsiab lus tseem ceeb ntawm kev xaiv cov tshuaj lysis yog xav txog ua ke nrog txoj kev ua kom huv.Muaj ib qho kev zam:Cov qauv uas muaj cov ntsiab lus endogenous enzyme tau pom zoo kom siv cov lysate uas muaj phenol kom muaj peev xwm ua kom tsis muaj zog endogenous enzymes.
Xaiv txoj kev purification
Cov yam ntxwv uas cuam tshuam rau cov seem endogenous impurities, extraction ceev Rau cov qauv huv xws li cov hlwb, cov txiaj ntsig zoo tuaj yeem tau txais nrog yuav luag txhua txoj kev ua kom huv ntawm tes.Tab sis rau ntau lwm cov qauv, tshwj xeeb tshaj yog cov uas muaj siab impurities xws li cov nroj tsuag, daim siab, kab mob, thiab lwm yam, xaiv ib tug haum purification txoj kev yog tseem ceeb heev.Cov txheej txheem centrifugal purification kem muaj qhov ceev ceev ceev thiab tuaj yeem tshem tawm cov impurities uas cuam tshuam rau cov tshuaj tiv thaiv enzymatic tom qab ntawm RNA, tab sis nws kim heev (Foregene tuaj yeem muab cov khoom siv tau zoo, ntau cov ntsiab lus nyemntawm no);siv kev lag luam thiab kev ua kom huv huv classic, xws li LiCl nag lossis daus, tuaj yeem tau txais txiaj ntsig zoo, tab sis lub sijhawm ua haujlwm ntev..
"Peb Txoj Kev Cai thiab Yim Lub Cev" rau RNA Extraction
Kev qhuab ntuas 1:Muab xaus rau kev sib kis ntawm exogenous enzymes.
Nco tseg 1:Ua tib zoo hnav lub qhov ncauj qhov ntswg thiab hnab looj tes.
Lus Cim 2:Cov raj centrifuge, Tip taub hau, pipette rods, electrophoresis tso tsheb hlau luam, thiab kev sim cov rooj zaum koom nrog hauv qhov kev sim yuav tsum muab pov tseg kom huv si.
Lus Cim 3:Cov reagents / kev daws teeb meem koom nrog hauv kev sim, tshwj xeeb tshaj yog dej, yuav tsum yog RNase-dawb.
Kev qhuab ntuas 2:Thaiv cov haujlwm ntawm endogenous enzymes
Lus Cim 4:Xaiv ib txoj kev tsim nyog homogenization.
Lus Cim 5:Xaiv ib qho lysate tsim nyog.
Lus Cim 6:Tswj tus nqi pib ntawm tus qauv.
Kev qhuab ntuas 3:Qhia meej koj lub hom phiaj extraction
Lus Cim 7:Nrog rau ib qho lysate system mus txog qhov siab tshaj plaws pib tus qauv, qhov kev rho tawm qhov kev vam meej poob qis heev.
Lus Cim 8:Tib qho kev ntsuas kev lag luam rau kev ua tiav RNA rho tawm yog kev ua tiav hauv kev sim tom ntej, tsis yog tawm los.
Sab saum toj 10 Qhov Chaw ntawm RNase Contamination
1. Cov ntiv tes yog thawj qhov chaw ntawm exogenous enzymes, yog li cov hnab looj tes yuav tsum tau hnav thiab hloov ntau zaus.Tsis tas li ntawd, lub qhov ncauj qhov ntswg kuj yuav tsum tau hnav, vim tias kev ua pa kuj yog ib qho tseem ceeb ntawm cov enzymes.Ib qho txiaj ntsig ntxiv ntawm kev hnav lub npog ntsej muag yog los tiv thaiv tus neeg sim.
2. Pipette cov lus qhia, cov raj centrifuge, pipettes - RNase tsis tuaj yeem ua tsis tau los ntawm kev ua kom tsis muaj menyuam ib leeg, yog li cov lus qhia pipette thiab cov raj centrifuge yuav tsum tau kho nrog DEPC, txawm tias lawv raug cim raws li DEPC kho.Nws yog qhov zoo tshaj plaws los siv lub hom phiaj tshwj xeeb pipette, so nws nrog 75% cawv paj rwb pob ua ntej siv, tshwj xeeb tshaj yog tus pas nrig;Tsis tas li ntawd, nco ntsoov tsis txhob siv lub taub hau tshem tawm.
3. Cov dej / tsis yuav tsum tsis muaj RNase paug.
4. Yam tsawg kawg ntawm lub rooj xeem yuav tsum tau muab ntxuav kom huv nrog 75% cawv paj rwb pob.
5.Endogenous RNase Tag nrho cov ntaub so ntswg muaj endogenous enzymes, yog li ceev ceev khov ntawm cov ntaub so ntswg nrog kua nitrogen yog txoj kev zoo tshaj plaws los txo cov degradation.Cov kua nitrogen cia / sib tsoo txoj kev yog qhov tsis yooj yim, tab sis nws yog tib txoj hauv kev rau cov ntaub so ntswg nrog qib siab ntawm endogenous enzymes.
6. RNA cov qauv RNA rho tawm cov khoom yuav muaj cov kab mob RNase.
7. Plasmid extraction Plasmid extraction feem ntau siv Rnase kom degrade RNA, thiab cov seem Rnase yuav tsum tau zom nrog Proteinase K thiab muab rho tawm los ntawm PCI.
8. RNA cia Txawm tias nws khaws cia ntawm qhov kub thiab txias, cov kab ntawm RNase yuav ua rau RNA degradation.Txoj kev daws teeb meem zoo tshaj plaws rau kev khaws cia ntev ntev ntawm RNA yog ntsev / cawv, vim tias cawv inhibits tag nrho cov enzymatic kev ua haujlwm ntawm qhov kub thiab txias.
9. Thaum cations (Ca, Mg) muaj cov ions, cua sov ntawm 80C rau 5 feeb yuav ua rau RNA cleaved, yog li yog hais tias RNA yuav tsum tau rhuab, lub preservation tov yuav tsum muaj ib tug chelate agent (1mM Sodium Citrate, pH 6.4).
10. Cov enzymes uas siv rau hauv kev sim tom ntej yuav kis tau los ntawm RNase.
10 Lub Tswv Yim rau RNA Extraction
1: Ceev ceev tiv thaiv RNase kev ua.Cov qauv yog khov sai sai tom qab sau, thiab RNase yog inactivated los ntawm kev khiav hauj lwm ceev thaum lub sij hawm lysis.
2: Xaiv ib txoj kev rho tawm tsim nyog rau cov ntaub so ntswg nrog cov ntsiab lus ribozyme siab, thiab cov ntaub so ntswg adipose yog qhov zoo tshaj plaws los siv txoj kev uas muaj phenol.
3: Prediction zoo yuav tsum tau Northern, cDNA tsev qiv ntawv kev tsim kho yuav tsum muaj siab ncaj ncees, thiab RT-PCR thiab RPA (Ribonuclease tiv thaiv assay) tsis xav tau siab ncaj ncees.RT-PCR yuav tsum muaj siab purity (enzyme inhibitor residues).
4: Kev sib xyaw ua ke zoo yog tus yuam sij rau kev txhim kho cov txiaj ntsig thiab txo qis degradation.
5: Tshawb xyuas qhov kev ntseeg siab ntawm RNA electrophoresis nrhiav kom pom, 28S: 18S = 2: 1 yog ib qho kev ua tiav, 1: 1 kuj tseem siv tau rau ntau qhov kev sim.
6: Tshem tawm DNA rau RT-PCR, array tsom xam Nws yog qhov zoo tshaj plaws los siv Dnase I tshem DNA.
7: Txo cov kab mob ntawm exogenous enzymes - enzymes tsis tuaj yeem raug xa tawm los ntawm sab nraud.
8: Thaum concentrating low-concentration nucleic acid, ib co-precipitation reagent yuav tsum tau ntxiv.Tab sis kom tiv thaiv cov co-precipitant uas muaj enzymes thiab DNA paug.
9: Ua kom tiav RNA, yog tias tsim nyog, kub ntawm 65C rau 5 feeb.
tsim nyog txoj kev cia
Nws tuaj yeem khaws cia ntawm -20C rau lub sijhawm luv, thiab ntawm -80C rau lub sijhawm ntev.Thawj kauj ruam hauv kev txhim kho RNA cov txiaj ntsig yog kom paub tias RNA cov ntsiab lus ntawm cov qauv sib txawv txawv heev.Muaj ntau ntau (2-4ug/mg) xws li daim siab, txiav, lub plawv, nruab nrab abundance (0.05-2ug/mg) xws li lub paj hlwb, embryo, raum, ntsws, thymus, zes qe menyuam, tsawg abundance (<0.05ug/mg) mg) xws li zais zis, pob txha, rog.
1: Lyse hlwb kom tso tawm RN - yog tias RNA tsis tso tawm, cov txiaj ntsig yuav raug txo.Hluav taws xob homogenization ua haujlwm zoo dua li lwm txoj hauv kev homogenization, tab sis kuj yuav tsum tau ua ke nrog lwm txoj kev, xws li kua nitrogen mashing, enzymatic digestion (Lysozyme / Lyticase)
2: Optimization ntawm txoj kev rho tawm.Cov teeb meem loj tshaj plaws nrog phenol-raws li txoj hauv kev yog tsis tiav stratification thiab ib nrab RNA poob (lub supernatant tsis tuaj yeem tshem tawm tag nrho).Kev ua tsis tiav stratification yog vim muaj cov nucleic acid siab thiab cov ntsiab lus protein, uas tuaj yeem daws tau los ntawm kev nce cov lysate siv los yog txo tus nqi ntawm cov qauv.Ib kauj ruam ntawm chloroform extraction tau ntxiv rau cov ntaub so ntswg adipose.RNA poob tuaj yeem raug txo los ntawm rov qab-tso los yog tshem tawm cov organic txheej ua raws li centrifugation.Qhov teeb meem loj tshaj plaws nrog kab centrifugation-raws li txoj hauv kev yog cov qauv ntau dhau.
Classic Extraction Tips
1. Phenol purification: Ntxiv ib qho sib npaug ntawm 1: 1 Phenol / Chloroform thiab sib xyaw ua ke rau 1-2 feeb.Centrifuge ntawm kev kub ceev rau 2 feeb.Ua tib zoo tshem tawm cov supernatant (80-90%).Tsis txhob mus rau nruab nrab txheej.Ib qho sib npaug ntawm cov tshuaj tiv thaiv tuaj yeem muab ntxiv rau Phenol / Chloroform thiab cov supernatant tshem tawm.Ob lub supernatants tuaj yeem sib xyaw ua ke rau nucleic acid nag lossis daus los txhim kho cov txiaj ntsig.Tsis txhob maj maj thaum sib tov, thiab tsis txhob sim tshem tag nrho cov supernatant.
2. Ntxuav nrog 70-80% ethanol: Thaum lub sij hawm ntxuav, cov nucleic acid yuav tsum tau muab tshem tawm kom ntseeg tau tias cov ntsev seem yog ntxuav tawm.Nyob rau tib lub sijhawm, tam sim ntawd tom qab nchuav tawm ntawm ethanol, centrifuge ntawm kev kub ceev rau ob peb feeb, thiab tom qab ntawd tshem tawm cov seem ethanol nrog lub pipette.Dissolve tom qab sawv ntawm chav tsev kub rau 5-10 feeb.
11. Kev rho tawm cov koom haum tshwj xeeb
1. Fibrous cov ntaub so ntswg: Tus yuam sij rau RNA rho tawm los ntawm cov ntaub so ntswg xws li lub plawv/skeletal leeg yog ua kom tag nrho cov ntaub so ntswg.Cov ntaub so ntswg muaj tsawg ntawm tes, yog li tus nqi ntawm RNA ib chav tsev qhov hnyav ntawm cov ntaub so ntswg yog tsawg, thiab nws yog qhov zoo tshaj plaws los siv ntau npaum li qhov pib tau.Nco ntsoov zom cov ntaub so ntswg kom huv si nyob rau hauv qhov chaw khov.
2. Cov ntaub so ntswg uas muaj cov protein ntau / rog: lub hlwb / cov zaub muaj roj ntau.Tom qab PCI rho tawm, cov supernatant muaj cov floccules dawb.Lub supernatant yuav tsum tau muab rho tawm nrog chloroform.
3. Cov ntaub so ntswg uas muaj cov ntsiab lus nucleic acid / ribozyme: tus po / thymus muaj cov nucleic acid siab thiab cov ntsiab lus ribozyme.Kev sib tsoo cov ntaub so ntswg nyob rau hauv qhov txias txias ua raws li homogenization sai tuaj yeem ua kom tsis muaj zog ribozymes.Txawm li cas los xij, yog tias cov lysate hnyav dhau lawm (vim muaj cov ntsiab lus nucleic acid siab), PCI extraction yuav tsis muaj peev xwm stratify zoo;ntxiv lysate ntau tuaj yeem daws qhov teeb meem no.Ntau PCI rho tawm tuaj yeem tshem tawm DNA seem ntxiv.Yog tias daim ntawv dawb precipitate tam sim ntawd tom qab ntxiv cawv, nws qhia tias muaj kab mob DNA.Kev rho tawm nrog cov kua qaub PCI tom qab kev sib cais tuaj yeem tshem tawm cov kab mob DNA.
4. Cov ntaub so ntswg: cog cov ntaub so ntswg ntau dua li cov ntaub so ntswg.Feem ntau, cov nroj tsuag yog av nyob rau hauv cov kua nitrogen, yog li RNA degradation los ntawm endogenous enzymes yog tsis tshua muaj.Yog tias qhov teeb meem degradation tsis daws, nws yuav luag yog tshwm sim los ntawm impurities muaj nyob hauv cov qauv.Cov impurities muaj nyob rau hauv ntau cov nroj tsuag yuav ua rau residues, thiab yog vim li cas rau residues feem ntau yog vim cov impurities muaj ib co zoo sib xws nrog RNA: koj precipitate thiab kuv precipitate, thiab koj adsorb thiab kuv adsorb.Cov yam ntxwv no txiav txim siab tias lawv yog cov muaj zog enzyme inhibitors.
Tam sim no, kev lag luam RNA extraction reagents tuaj yeem hloov mus rau yuav luag tag nrho cov tsiaj cov ntaub so ntswg nrog kev hloov me me, tab sis muaj qee qhov kev lag luam RNA extraction reagents uas tsim nyog rau feem ntau cov ntaub so ntswg.Hmoov zoo, Foregene tuaj yeem muab tshwj xeebcog RNA cov khoom siv rho tawm, peb muajPlant Total RNA Isolation Kit, Plant Total RNA Isolation Kit Plus.Cov tom kawg yog tsim tshwj xeeb rau cov nroj tsuag nrog siab polysaccharide thiab polyphenol cov ntsiab lus.Rau RNA rho tawm, tawm tswv yim los ntawm cov neeg siv lab yog qhov zoo tshwj xeeb.
12. Cov nyhuv ntawm cov qauv khov thiab thawing Cov qauv khov yuav loj dua, thiab nws yuav tsum tau txiav ua ntej siv rau RNA extraction.Cov qauv zoo li yaj (tej zaum ib nrab) thaum txiav.Cov qauv khov yuav tsum tau ntsuas ua ntej RNA rho tawm, thiab thawing yuav tshwm sim thaum lub sijhawm txheej txheem no.Qee zaum, thawing ntawm cov qauv kuj tshwm sim thaum lub sij hawm ua kua nitrogen milling txheej txheem;los yog cov qauv khov yog ncaj qha ntxiv rau lysate yam tsis muaj kua nitrogen milling, thiab thawing yuav tshwm sim ua ntej ua tiav homogenization.Cov kev sim tau pom tias cov ntaub so ntswg khov muaj ntau dua rau RNA degradation thaum thawing dua li cov ntaub so ntswg tshiab.Qhov laj thawj zoo: Cov txheej txheem khov-thaw cuam tshuam cov qauv hauv lub cell, ua kom yooj yim rau cov enzymes endogenous tuaj rau hauv kev sib cuag ncaj qha nrog RNA.
13. Kev txiav txim siab ntawm RNA zoo Feem ntau, electrophoresis yog siv los txiav txim qhov ncaj ncees ntawm RNA, thiab A260 / A280 yog siv los txiav txim qhov purity ntawm RNA.Hauv txoj kev xav, qhov tsis zoo RNA muaj qhov sib piv ntawm 28S: 18S = 2.7: 1, thiab cov ntaub ntawv feem ntau hais txog qhov piv ntawm 28S: 18S = 2: 1.Qhov tseeb yog tias yuav luag tsis muaj ib qho ntawm RNA rho tawm los ntawm cov qauv uas tsis yog cov hlwb yog nyob rau hauv 2: 1 piv (qhov no tau txais los ntawm Agilent Bioanalyzer).
Cov txiaj ntsig ntawm electrophoresis ntawm RNA cuam tshuam los ntawm ntau yam, suav nrog cov qauv theem nrab, electrophoresis tej yam kev mob, qauv load, degree ntawm saturation los ntawm EB, thiab lwm yam. Siv haiv neeg electrophoresis los ntes RNA thiab siv DNA Marker los tswj.Yog tias 28S ntawm 2kb thiab 18S ntawm 0.9kb yog qhov tseeb, thiab 28S: 18S> 1, kev ncaj ncees tuaj yeem ua tau raws li qhov yuav tsum tau ua ntawm feem ntau cov kev sim tom ntej.
A260 / A280 yog qhov taw qhia uas tau ua rau muaj kev ntxhov siab ntau.Ua ntej tshaj plaws, nws yog ib qho tsim nyog los qhia meej lub ntsiab lus tseem ceeb ntawm qhov ntsuas no rau nucleic acids: ntshiab RNA, nws A260/280 = txog 2.0.Ntshiab RNA yog qhov 'ua rau' thiab A260 / A280 = 2 yog 'kev cuam tshuam'.Tam sim no txhua tus tau siv A260 / A280 ua 'ua rau', xav tias "yog A260 / A280 = 2, ces RNA yog ntshiab", uas ib txwm ua rau tsis meej pem.
Yog tias koj txaus siab, koj tuaj yeem ntxiv me ntsis reagent uas feem ntau siv hauv kev rho tawm, xws li phenol, guanidine isothiocyanate, PEG, thiab lwm yam, rau koj tus qauv RNA, thiab tom qab ntawd ntsuas qhov A260 / A280 piv.Qhov tseeb yog tias ntau cov reagents siv rau RNA rho tawm, nrog rau ntau yam impurities hauv cov qauv, nqus ib ncig ntawm A260 thiab A280, cuam tshuam rau A260 / A280.
Txoj kev qhia tshaj plaws tam sim no yog txhawm rau kuaj RNA cov qauv hauv 200-300 nm ntau.Qhov nkhaus ntawm RNA ntshiab muaj cov yam ntxwv hauv qab no: qhov nkhaus yog du, A230 thiab A260 yog ob lub ntsiab lus inflection, A300 yog ze rau 0, A260 / A280 = nyob ib ncig ntawm 2.0, thiab A260 / A230 = nyob ib ncig ntawm 2.0.Yog tias tsis muaj cov ntaub ntawv scan, A260 / A230 piv yuav tsum tau txiav txim siab, vim qhov piv txwv no muaj kev cuam tshuam ntau dua rau kev nqa ntawm txhua qhov impurities uas cuam tshuam rau cov tshuaj tiv thaiv enzymatic.Coj mus rau hauv tus account lub linear ntau ntawm cov cuab yeej (0.1-0.5 rau A260).
Muaj ob qhov kev tshwm sim muaj txiaj ntsig: qhov piv yuav yog li 0.3 qis dua thaum A260 / A280 ntsuas hauv dej;thaum qhov piv ntsuas hauv 10 mM EDTA yog li 0.2 siab dua qhov ntsuas hauv 1 mM EDTA.
Yam khoom:
Tuam Tshoj Cog Tag Nrho RNA Isolation Kit Chaw tsim tshuaj paus thiab tsum |Foregene (foreivd.com)
RNA cais tawm series - Foregene Co., Ltd. (foreivd.com)
Post lub sij hawm: Lub Xya hli ntuj-15-2022
