Kutsekereza malangizo a pipette ndi machubu a EP, ndi zina.
1. Konzani 0.1% (chikwi chimodzi) DEPC (chinthu choopsa kwambiri) ndi madzi osungunuka, mugwiritseni ntchito mosamala mu fume hood, ndikuyisunga pa 4 ° C kutali ndi kuwala;
Madzi a DEPC ndi madzi oyera oyeretsedwa ndi DEPC ndipo amatsukidwa ndi kutentha kwambiri komanso kuthamanga kwambiri.Anayesedwa kuti alibe RNase, DNase ndi proteinase.
2. Ikani nsonga ya pipette ndi EP chubu mu 0.1% DEPC, ndipo onetsetsani kuti nsonga ya pipette ndi EP chubu zimadzazidwa ndi 0.1% DEP.
3. Tetezani ku kuwala, tiyeni tiyime usiku wonse (12-24h)
4. Bokosi lomwe lili ndi nsonga ndi chubu la EP siliyenera kulowetsedwa mu DEPC.Pambuyo pochotsa pafupifupi madzi a DEPC pansonga kapena chubu la EP, nyamulani ndikukulunga.
5. 121 digiri Celsius, 30min
6. 180 digiri Celsius, zouma kwa maola angapo (osachepera 3 hours)
Chidziwitso: a.Valani magolovesi a latex ndi masks mukamagwira DEPC!b, kapena popanda kutsekereza kwa DEPC, 130 ℃, 90min autoclave (ma laboratories ambiri kutentha kutsekereza kawiri)
Malingaliro a magawo a RNA
Zochitika ziwiri zazikulu za minofu RNA kudzipatula kulephera
Kuwonongeka kwa RNA ndi zotsalira za zonyansa mu minofu,ponena za kuwonongeka, tiyeni tiyang'ane kaye chifukwa chake RNA yotengedwa ku maselo otukuka siiwonongeka mosavuta.Zomwe zilipo kale za RNA zotulutsa zonse zili ndi zigawo zomwe zimalepheretsa RNase mwachangu.Onjezani lysate ku maselo otukuka, ndikungosakaniza, maselo onse akhoza kusakanikirana bwino ndi lysate, ndipo maselo amasungunuka kwathunthu.Maselo atatha lysed, zosakaniza zogwira ntchito mu lysate nthawi yomweyo zimalepheretsa RNase intracellular, kotero RNA imakhalabe.Ndiko kunena kuti, chifukwa maselo otukuka amalumikizana mosavuta ndi lysate, RNA yawo siwonongeka mosavuta;Komano, RNA mu minofu imawonongeka mosavuta chifukwa maselo mu minofu sizovuta kuti agwirizane ndi lysate mwamsanga.chifukwa chokhudzana mokwanira.Choncho,poganiza kuti pali njira yosinthira minofu kukhala selo limodzi ndikuletsa ntchito ya RNA, vuto la kuwonongeka likhoza kuthetsedwa kwathunthu.
Mphero ya nayitrogeni yamadzimadzi ndiyo njira yothandiza kwambiri.Komabe, njira ya mphero ya nayitrogeni yamadzimadzi ndiyovuta kwambiri, makamaka ngati kuchuluka kwa zitsanzo kuli kwakukulu.Izi zinayambitsa chinthu chotsatira chabwino: homogenizer.ThehomogenizerNjira saganiziranso funso la momwe ntchito ya RNase imaletsedwera maselo asanakumane ndi lysate, koma amapemphera kuti kusokonezeka kwa minofu kumathamanga kwambiri kuposa momwe intracellular RNase imawonongera RN.
Zotsatira za homogenizer yamagetsi ndizabwinoko,ndipo zotsatira za galasi homogenizer ndi osauka, koma ambiri, njira homogenizer sangathe kuletsa chodabwitsa kudzitsitsa.Choncho, ngati m'zigawo ndi oipa, choyambirira homogenizer magetsi ayenera kugwiritsidwa ntchito akupera ndi madzi asafe;homogenizer yagalasi yoyambirira iyenera kusinthidwa kukhala homogenizer yamagetsi kapena milled mwachindunji ndi nayitrogeni wamadzimadzi.Vuto ndi pafupifupi 100% zotheka.kuthetsedwa.
Vuto la zotsalira za zonyansa lomwe limakhudza zoyeserera zotsatila liri ndi zifukwa zosiyanasiyana kuposa kuwonongeka, ndipo mayankho ake ndi osiyana.Pomaliza,ngati pali kuwonongeka kapena zonyansa zotsalira mu minofu, njira yotulutsira / yopangira zinthu zenizeni zoyesera ziyenera kukonzedwa.Simuyenera kugwiritsa ntchito zitsanzo zanu zamtengo wapatali kuti muthe kukhathamiritsa: mutha kugula nyama zazing'ono monga nsomba / nkhuku kuchokera kumsika, kutenga gawo lofananira lazinthu zochotsa RNA, ndi gawo lina lochotsa mapuloteni - perani ndi pakamwa, m'mimba ndi matumbo.
Cholinga cha RNA cha RNA yochotsedwa chimagwiritsidwa ntchito pazoyeserera zosiyanasiyana zotsatiridwa, ndipo zofunikira zake zamtundu ndizosiyana
Kumanga laibulale ya cDNA kumafuna kukhulupirika kwa RNA popanda zotsalira za enzyme reaction inhibitors;Kumpoto kumafuna umphumphu wapamwamba wa RNA ndi zofunikira zochepa za zotsalira za enzyme reaction inhibitors;RT-PCR sichifuna kukhulupirika kwambiri kwa RNA,koma amalepheretsa ma enzyme.Zofunikira zotsalira ndizokhwima.Zomwe zimalowetsa zimatsimikizira zotuluka;nthawi iliyonse cholinga ndikupeza chiyero chapamwamba kwambiri cha RNA, chidzawononga anthu ndi ndalama.
Kutolera/Kusunga Zitsanzo
Zomwe Zimakhudza Kuwonongeka Pambuyo sampuli ikachoka m'thupi lamoyo / kapena malo omwe amakulirakulira, ma enzyme omwe amapezeka pachitsanzo amayamba kusokoneza RNA,ndipo kuchuluka kwa kuwonongeka kumakhudzana ndi zomwe zili m'matumbo am'mimba komanso kutentha.Pachikhalidwe, pali njira ziwiri zokha zoletsera amkati enzyme ntchito: kuwonjezera lysate yomweyo ndi homogenize bwinobwino ndi mofulumira;kudula mutizidutswa tating'ono ting'ono ndipo nthawi yomweyo amaundana mu madzi asafe.Njira ziwirizi zimafuna kugwira ntchito mwachangu.Yotsirizira ndi oyenera onse zitsanzo, pamene woyamba ndi oyenera zimakhala ndi otsika zili maselo ndi amkati michere ndi zosavuta homogenize.Makamaka, minofu ya zomera, chiwindi, thymus, kapamba, ndulu, ubongo, mafuta, minofu ya minofu, ndi zina zotero ndizozizira kwambiri ndi nayitrogeni wamadzimadzi musanapitirize.
Kugawikana ndi homogenization wa zitsanzo
Zomwe Zimakhudza Kuwonongeka ndi Kugawanika kwa Zitsanzo ndikwa homogenization mokwanira, yomwe ndi yomasulira kwathunthu ndi kotheratu kwa RNA.Maselo akhoza mwachindunji homogenized popanda wosweka.Matupi amatha kukhala homogenized pokhapokha atasweka.Yisiti ndi mabakiteriya ayenera kuthyoledwa ndi michere yofananira asanayambe kukhala homogenized.Minofu ndi m'munsi amkati enzyme zili ndi mosavuta homogenization akhoza wosweka ndi homogenized nthawi imodzi mu lysate ndi homogenizer;minyewa yamafuta, chiwindi, thymus, kapamba, ndulu, ubongo, mafuta, minofu ndi zitsanzo zina, mwina zimakhala ndi ma enzymes am'mimba kapena sizipanga homogenized,kotero kusokonezeka kwa minofu ndi homogenization ziyenera kuchitidwa mosiyana.Njira yodalirika komanso yopindulitsa kwambiri yogawanika ndi mphero ndi nayitrogeni wamadzimadzi, ndipo njira yodalirika kwambiri yopangira homogenization ndikugwiritsa ntchito homogenizer yamagetsi.Chidziwitso chapadera chokhudza mphero ndi nayitrogeni wamadzimadzi: chitsanzocho sichiyenera kusungunuka panthawi yonse ya mphero, chifukwa ma enzyme amkati amatha kugwira ntchito atazizira.
Kusankha kwa lysate
Kukhudza kumasuka kwa ntchito ndi zinthu zotsalira amkati zonyansa The ambiri ntchito njira lysis akhoza pafupifupi ziletsa ntchito ya RNase.Choncho, mfundo yofunika kwambiri posankha njira yothetsera lysis ndiyo kuganizira mophatikizana ndi njira yoyeretsera.Pali chinthu chimodzi chokha:zitsanzo ndi mkulu amkati enzyme zili tikulimbikitsidwa kugwiritsa ntchito lysate munali phenol kuonjezera luso inactivate amkati michere.
Kusankha njira yoyeretsera
Zinthu zomwe zimakhudza zotsalira zotsalira zamkati, kuthamanga kwa m'zigawo Kwa zitsanzo zoyera monga maselo, zotsatira zokhutiritsa zitha kupezeka ndi njira iliyonse yoyeretsera yomwe ili pafupi.Koma kwa zitsanzo zina zambiri, makamaka zomwe zili ndi zonyansa zambiri monga zomera, chiwindi, mabakiteriya, ndi zina zotero, kusankha njira yoyenera yoyeretsera ndikofunikira.Njira yoyeretsera gawo la centrifugal imakhala ndi liwiro lotulutsa mwachangu ndipo imatha kuchotsa bwino zonyansa zomwe zimakhudza ma enzymatic reaction ya RNA, koma ndizokwera mtengo (Forgene atha kupereka zida zotsika mtengo, zambiri dinaniPano);kugwiritsa ntchito njira zoyeretsera zachuma komanso zapamwamba, monga mvula ya LiCl, zithanso kupeza zotsatira zokhutiritsa, koma nthawi yogwira ntchito ndi yayitali..
"Malangizo Atatu ndi Zisanu ndi zitatu" za RNA Extraction
Chilango 1:Kuthetsa kuipitsidwa kwa exogenous michere.
Chidziwitso 1:Valani mwamphamvu masks ndi magolovesi.
Chidziwitso 2:Machubu a centrifuge, mitu ya nsonga, ndodo za pipette, akasinja a electrophoresis, ndi mabenchi oyesera omwe akukhudzidwa ndi kuyesa ayenera kutayidwa bwino.
Chidziwitso 3:Ma reagents/mayankho omwe akukhudzidwa pakuyesa, makamaka madzi, ayenera kukhala opanda RNase.
Chilango 2:Kuletsa ntchito amkati michere
Chidziwitso 4:Sankhani njira yoyenera ya homogenization.
Chidziwitso 5:Sankhani lysate yoyenera.
Chidziwitso 6:Lamulirani kuchuluka koyambira kwachitsanzo.
Chilango 3:Fotokozani cholinga chanu chochotsa
Chidziwitso 7:Ndi dongosolo lililonse la lysate likuyandikira kuchuluka koyambira kwachitsanzo, chiwopsezo cham'zigawo chimatsika kwambiri.
Chidziwitso 8:Njira yokhayo yachuma yochotsera bwino RNA ndikupambana pazoyeserera, osati zokolola.
Magwero 10 Opambana a Kuwonongeka kwa RNase
1. Zala ndizo gwero loyamba la ma enzymes akunja, kotero magolovesi amayenera kuvalidwa ndikusinthidwa pafupipafupi.Kuphatikiza apo, masks ayeneranso kuvala, chifukwa kupuma ndi gawo lofunikira la michere.Ubwino wowonjezera wovala chigoba cha glove ndikuteteza woyesera.
2. Malangizo a pipette, machubu a centrifuge, pipettes - RNase sangathe kutsekedwa ndi kutseketsa kokha, kotero nsonga za pipette ndi machubu a centrifuge ayenera kuchitidwa ndi DEPC, ngakhale atalembedwa ngati DEPC.Ndi bwino kugwiritsa ntchito pipette yopangidwa ndi cholinga chapadera, pukutani ndi 75% mowa wa thonje mpira musanagwiritse ntchito, makamaka ndodo;kuonjezerapo, onetsetsani kuti musagwiritse ntchito chochotsa mutu.
3. Madzi/bafa akuyenera kukhala opanda kuipitsidwa kwa RNase.
4. Osachepera tebulo loyesera liyenera kupukutidwa ndi mipira ya thonje ya 75% ya mowa.
5.Endogenous RNase Minofu yonse imakhala ndi ma enzymes amkati, kotero kuzizira kofulumira kwa minyewa yokhala ndi nayitrogeni yamadzi ndiyo njira yabwino yochepetsera kuwonongeka.Njira yosungiramo nayitrogeni yamadzimadzi ndiyovuta, koma ndi njira yokhayo ya minofu yokhala ndi ma enzymes ambiri.
6. RNA zitsanzo RNA m'zigawo mankhwala angakhale ndi kuda RNase kuipitsidwa.
7. Kutulutsa kwa Plasmid Kutulutsa kwa Plasmid nthawi zambiri kumagwiritsa ntchito Rnase kusokoneza RNA, ndipo Rnase yotsalira iyenera kugayidwa ndi Proteinase K ndikuchotsedwa ndi PCI.
8. Kusungidwa kwa RNA Ngakhale kusungidwa kutentha kochepa, kufufuza kuchuluka kwa RNA kungayambitse kuwonongeka kwa RNA.Njira yabwino yothetsera kusungidwa kwa nthawi yaitali kwa RNA ndi kuyimitsidwa kwa mchere / mowa, chifukwa mowa umalepheretsa ntchito zonse za enzymatic pa kutentha kochepa.
9. Pamene ma cations (Ca, Mg) ali ndi ma ions awa, kutentha kwa 80C kwa mphindi 5 kumapangitsa kuti RNA iwonongeke, choncho ngati RNA iyenera kutenthedwa, njira yotetezera iyenera kukhala ndi chelating agent (1mM Sodium Citrate, pH 6.4).
10. Ma enzyme omwe amagwiritsidwa ntchito poyesa kotsatira akhoza kuipitsidwa ndi RNase.
Malangizo 10 a RNA M'zigawo
1: Chepetsani ntchito ya RNase mwachangu.Zitsanzo zimayimitsidwa mwamsanga pambuyo posonkhanitsa, ndipo RNase imatsekedwa ndi ntchito yofulumira panthawi ya lysis.
2: Sankhani yoyenera m'zigawo njira minofu ndi mkulu ribozyme okhutira, ndi adipose minofu ndi bwino ntchito njira munali phenol.
3: Ubwino wolosera umafunika Kumpoto, kumanga laibulale ya cDNA kumafuna kukhulupirika kwakukulu, ndipo RT-PCR ndi RPA (Ribonuclease protection assay) sizifuna kukhulupirika kwakukulu.RT-PCR imafuna kuyeretsedwa kwakukulu (zotsalira za enzyme inhibitor).
4: Homogenization mokwanira ndiye chinsinsi chothandizira zokolola komanso kuchepetsa kuwonongeka.
5: Yang'anani kukhulupirika kwa RNA electrophoresis kuzindikira, 28S: 18S = 2: 1 ndi chizindikiro chathunthu, 1: 1 imavomerezedwanso pazoyesera zambiri.
6: Kuchotsedwa kwa DNA kwa RT-PCR, kusanthula mndandanda Ndi bwino kugwiritsa ntchito Dnase I kuchotsa DNA.
7: Chepetsani kuipitsidwa kwa michere yakunja - ma enzyme sangathe kutumizidwa kunja.
8: Poika ma nucleic acid otsika kwambiri, co-precipitation reagent iyenera kuwonjezeredwa.Koma kuteteza co-precipitant munali michere ndi DNA kuipitsidwa.
9: Sungunulani bwino RNA, ngati kuli kofunikira, kutentha pa 65C kwa mphindi zisanu.
njira yoyenera yosungirako
Itha kusungidwa pa -20C kwakanthawi kochepa, komanso pa -80C kwa nthawi yayitali.Gawo loyamba pakuwongolera zokolola za RNA ndikuzindikira kuti zomwe zili mu RNA zamitundu yosiyanasiyana zimasiyana kwambiri.Kuchuluka kwakukulu (2-4ug / mg) monga chiwindi, kapamba, mtima, kuchuluka kwapakati (0.05-2ug / mg) monga ubongo, mluza, impso, mapapo, thymus, ovary, otsika kwambiri (<0.05ug / mg) mg) monga chikhodzodzo, fupa, mafuta.
1: Maselo a Lyse kuti amasule RN - ngati RNA simasulidwa, zokolola zidzachepetsedwa.Magetsi a homogenization amagwira ntchito bwino kuposa njira zina zopangira homogenization, koma angafunikirenso kuphatikizidwa ndi njira zina, monga madzi a nitrogen mashing, enzymatic digestion (Lysozyme/Lyticase)
2: Kukhathamiritsa kwa njira yochotsera.Mavuto aakulu ndi njira zochokera ku phenol ndizosakwanira stratification ndi kutayika pang'ono kwa RNA (chapamwamba sichingachotsedwe kwathunthu).Kusakhazikika kosakwanira kumachitika chifukwa cha kuchuluka kwa nucleic acid ndi mapuloteni, omwe amatha kuthetsedwa powonjezera kuchuluka kwa ma lysate omwe amagwiritsidwa ntchito kapena kuchepetsa kuchuluka kwa zitsanzo.Gawo la kuchotsa chloroform linawonjezeredwa ku minofu ya adipose.Kutayika kwa RNA kumatha kuchepetsedwa ndi kupopera kumbuyo kapena kuchotsa organic wosanjikiza ndikutsatiridwa ndi centrifugation.Vuto lalikulu ndi njira zopangira centrifugation ndizowonjezera zitsanzo.
Classic m'zigawo Malangizo
1. Phenol kuyeretsedwa: Onjezerani voliyumu yofanana ya 1: 1 Phenol / Chloroform ndikusakaniza mwamphamvu kwa mphindi 1-2.Centrifuge pa liwiro lalikulu kwa mphindi 2.Chotsani mosamala zamatsenga (80-90%).Osafika pagawo lapakati.Voliyumu yofanana ya yankho la yankho litha kuwonjezeredwa ku Phenol/Chloroform ndi supernatant kuchotsedwa.Ma supernatants awiriwa amatha kusakanikirana kuti apange mpweya wa nucleic acid kuti zokolola ziwonjezeke.Musakhale ofatsa kwambiri posakaniza, ndipo musayese kuchotsa zonse zamatsenga.
2. Kutsuka ndi 70-80% ethanol: Pakutsuka, nucleic acid iyenera kuyimitsidwa kuti iwonetsetse kuti mchere wotsalira watsukidwa.Pa nthawi yomweyo, atangotsanulira Mowa, centrifuge pa liwiro mkulu kwa masekondi angapo, ndiyeno kuchotsa Mowa yotsalira ndi pipette.Sungunulani atayima pa firiji kwa mphindi 5-10.
11. Kuchotsa mabungwe apadera
1. Minofu ya Fibrous: Chinsinsi cha kuchotsa RNA kuchokera ku minofu ya fibrous monga mtima / chigoba minofu ndikusokoneza kwathunthu minofu.Minofu iyi imakhala ndi kachulukidwe kakang'ono ka maselo, kotero kuchuluka kwa RNA pa unit kulemera kwa minofu kumakhala kochepa, ndipo ndi bwino kugwiritsa ntchito kuchuluka koyambira momwe mungathere.Onetsetsani kuti mukupera minofu bwinobwino pansi pa nyengo yozizira.
2. Minofu yokhala ndi mapuloteni / mafuta ambiri: mafuta a mu ubongo / masamba ndi ochuluka.Pambuyo pochotsa PCI, supernatant imakhala ndi ma floccules oyera.Chowonjezeracho chiyenera kuchotsedwanso ndi chloroform.
3. Minofu yokhala ndi nucleic acid / ribozyme yambiri: ndulu / thymus imakhala ndi nucleic acid ndi ribozyme.Kupukuta minofu pansi pa kuzizira kotsatiridwa ndi homogenization mofulumira kumatha kuyambitsa ribozymes.Komabe, ngati lysate ndi viscous kwambiri (chifukwa cha kuchuluka kwa nucleic acid), kuchotsa kwa PCI sikungathe stratify bwino;kuwonjezera lysate kungathe kuthetsa vutoli.Ma PCI angapo amatha kuchotsa DNA yotsalira.Ngati mvula yoyera ipangika mutangowonjezera mowa, izi zikuwonetsa kuipitsidwa kwa DNA.Kuchotsanso ndi acidic PCI pambuyo pa kusungunuka kumatha kuchotsa kuipitsidwa kwa DNA.
4. Minofu ya zomera: Minofu ya zomera ndi yovuta kwambiri kuposa ya nyama.Nthawi zambiri, mbewu zimasiyidwa pansi pamikhalidwe ya nayitrogeni wamadzimadzi, kotero kuwonongeka kwa RNA ndi ma enzymes amkati sikwachilendo.Ngati vuto lowonongeka silinathetsedwa, limakhala chifukwa cha zonyansa zomwe zili mu chitsanzocho.Zonyansa zomwe zili muzomera zambiri zidzatsogolera ku zotsalira, ndipo chifukwa cha zotsalira nthawi zambiri zimakhala chifukwa chakuti zonyansazi zimakhala ndi zofanana ndi RNA: mumathamanga ndikuthamanga, ndipo mumakukondani ndikukukondani.Makhalidwewa amatsimikizira kuti ndi amphamvu kwambiri ma enzyme inhibitors.
Pakali pano, malonda a RNA otulutsa ma reagents amatha kusinthidwa pafupifupi pafupifupi mitundu yonse ya nyama yokhala ndi zosintha zazing'ono, koma pali ma reagents ochepa a RNA omwe amatha kukhala oyenera kuzinthu zambiri zamitengo.Mwamwayi, Foregene akhoza kupereka wapaderambewu za RNA m'zigawo, tili ndiPlant Total RNA Isolation kit, Plant Total RNA Isolation kit Plus.Zotsirizirazi zimapangidwira mwapadera zomera zomwe zimakhala ndi polysaccharide yambiri ndi polyphenol.Pakuchotsa kwa RNA, mayankho ochokera kwa ogwiritsa ntchito labu ndizabwino kwambiri.
12. Zotsatira za kuzizira kwachitsanzo ndi kusungunuka Chitsanzo chozizira chikhoza kukhala chachikulu, ndipo chiyenera kudulidwa chisanagwiritsidwe ntchito pochotsa RNA.Zitsanzo zimasungunuka (mwina pang'ono) panthawi yodula.Zitsanzo zozizira zingafunikire kuyezedwa asanatulutse RNA, ndipo kusungunuka kudzachitika panthawiyi.Nthawi zina, ndi thawing wa chitsanzo amapezekanso pa madzi asafe mphero;kapena mazira chitsanzo mwachindunji anawonjezera kwa lysate popanda madzi asafe mphero, ndi thawing zidzachitika pamaso wathunthu homogenization.Kuyesera kwawonetsa kuti minofu yowundana ndiyomwe imakonda kuwonongeka kwa RNA panthawi yosungunuka kuposa minofu yatsopano.Chifukwa chake n’chakuti: Kuzizira kozizira kumasokoneza zinthu zomwe zili mkati mwa selo, zomwe zimapangitsa kuti ma enzymes opezeka m’thupi agwirizane ndi RNA mosavuta.
13. Chiweruzo cha khalidwe la RNA Kawirikawiri, electrophoresis imagwiritsidwa ntchito poweruza kukhulupirika kwa RNA, ndipo A260 / A280 imagwiritsidwa ntchito kuweruza chiyero cha RNA.Mwachidziwitso, RNA yosasunthika ili ndi chiŵerengero cha 28S: 18S = 2.7: 1, ndipo deta yambiri imatsindika chiŵerengero cha 28S: 18S = 2: 1.Chowonadi ndi chakuti pafupifupi palibe RNA yotengedwa ku zitsanzo zina kupatula ma cell omwe ali mu chiŵerengero cha 2: 1 (izi zinapezedwa pogwiritsa ntchito Agilent Bioanalyzer).
Zotsatira za electrophoresis za RNA zimakhudzidwa ndi zinthu zambiri, kuphatikizapo dongosolo lachiwiri, electrophoresis mikhalidwe, katundu wa chitsanzo, kuchuluka kwa machulukitsidwe ndi EB, etc. Gwiritsani ntchito electrophoresis yachibadwa kuti mudziwe RNA ndikugwiritsa ntchito DNA Marker monga ulamuliro.Ngati 28S pa 2kb ndi 18S pa 0.9kb ndi zomveka, ndi 28S: 18S> 1, umphumphu akhoza kukwaniritsa zofunika za zoyesera zambiri wotsatira.
A260 / A280 ndi chizindikiro chomwe chayambitsa chisokonezo chachikulu.Choyamba, m'pofunika kufotokoza tanthauzo lenileni la chizindikiro ichi kwa nucleic zidulo: koyera RNA, A260/280 ake = pafupifupi 2.0.RNA yoyera ndiye 'chifukwa' ndipo A260/A280 = 2 ndiye 'zotsatira'.Tsopano aliyense akugwiritsa ntchito A260 / A280 monga 'chifukwa', kuganiza kuti "ngati A260 / A280 = 2, ndiye kuti RNA ndi yoyera", zomwe zimadzetsa chisokonezo.
Ngati mukufuna, mukhoza kuwonjezera reagent pang'ono kuti nthawi zambiri ntchito m'zigawo, monga phenol, guanidine isothiocyanate, PEG, etc., kuti RNA chitsanzo, ndiyeno kuyeza chiŵerengero A260/A280.Chowonadi ndi chakuti ma reagents ambiri omwe amagwiritsidwa ntchito pochotsa RNA, komanso zonyansa zambiri pachitsanzo, zimayamwa mozungulira A260 ndi A280, zomwe zimakhudza A260/A280.
Njira yophunzitsira kwambiri pakadali pano ndikusanthula zitsanzo za RNA mumtundu wa 200-300 nm.Mphepete mwa RNA yoyera ili ndi zizindikiro zotsatirazi: phirilo ndi losalala, A230 ndi A260 ndi mfundo ziwiri zolowera, A300 ili pafupi ndi 0, A260 / A280 = kuzungulira 2.0, ndi A260 / A230 = kuzungulira 2.0.Ngati scan data palibe, chiŵerengero cha A260/A230 chiyeneranso kutsimikiziridwa, chifukwa chiŵerengerochi chimakhala chokhudzidwa kwambiri ndi kunyamula zonyansa zonse zomwe zimakhudza momwe enzymatic reaction.Ganizirani za mzere wa chipangizocho (0.1-0.5 pa A260).
Palinso zochitika zina ziwiri zothandiza: chiŵerengerocho chidzakhala pafupi ndi 0,3 kutsika pamene A260 / A280 imayesedwa m'madzi;pamene chiŵerengero choyezedwa mu 10 mM EDTA chili pafupi ndi 0.2 kuposa chomwe chinayesedwa mu 1 mM EDTA.
Zogwirizana nazo:
China Plant Total RNA Isolation Kit wopanga ndi Supplier |Foregene (foreivd.com)
RNA kudzipatula mndandanda - Foregene Co., Ltd. (foreivd.com)
Nthawi yotumiza: Jul-15-2022
