ISityalo sisonke iKhithi yokuKwahlula yeRNA iyonke iRNA Purificaiton Kit yeSityalo esiPhantsi kwiiPolysaccharides kunye neePolyphenols
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Ikiti isebenzisa ikholomu ye-spin kunye nefomula ephuhliswe yi-Foregene, enokuthi ikhuphe ngokufanelekileyo ubunyulu obuphezulu kunye nomgangatho ophezulu we-RNA kwiintlobo ezahlukeneyo zezityalo ezine-polysaccharides eziphantsi kunye nomxholo we-polyphenols.Kwiisampuli zezityalo ezine-polysaccharides eziphezulu okanye umxholo we-polyphenols, kuyacetyiswa ukuba usebenzise i-Plant Total RNA Isolation Plus Kit ukuze ufumane iziphumo ezingcono ze-RNA zokukhutshwa.Ikiti inikezela nge-DNA-Cleaning column enokuthi isuse ngokulula i-DNA ye-genomic kwi-supernatant kunye ne-tissue lysate.Ikholamu ye-RNA kuphela inokubopha ngokufanelekileyo i-RNA.Ikhithi inokuqhuba inani elikhulu leesampuli ngexesha elinye.
Inkqubo yonke ayinayo i-RNase, ngoko ke i-RNA ecocekileyo ayiyi kuthotywa.I-Buffer PRW1 kunye ne-Buffer PRW2 inokuqinisekisa ukuba i-RNA efunyenweyo ayingcoliswanga yiprotheni, i-DNA, i-ion, kunye ne-organic compounds.
Amacandelo ekhithi
| Buffer PSL1, Buffer PS, Buffer PSL2 |
| Isithinteli PRW1, Isithinteli PRW2 |
| RNase-Free ddH2O, iKholamu yokucoca iDNA |
| Ikholamu ye-RNA Kuphela |
| Imiyalelo |
Iimpawu&izinto eziluncedo
■ Ukusebenza kwiqondo lobushushu begumbi (15-25℃) kuyo yonke inkqubo, ngaphandle kokuhlamba komkhenkce kunye nobushushu obuphantsi be-centrifugation.
■ Gqibezela ikhithi ye-RNase-Free, akukho mfuneko yakukhathazeka malunga nokuthotywa kwe-RNA.
∎ IKholam yokucoca i-DNA ibophelela ngokukodwa kwi-DNA, ukuze ikhithi ikwazi ukususa ungcoliseko lwe-genomic DNA ngaphandle kokongeza i-DNase.
■ Isivuno esiphezulu se-RNA: IKholamu ye-RNA kuphela kunye nefomula ekhethekileyo inokucoca ngokufanelekileyo i-RNA.
■ Isantya esikhawulezayo: kulula ukusebenza kwaye sinokugqitywa kwimizuzu engama-30.
■ Ukhuseleko: akukho sixhobo sokwenza izinto eziphilayo esifunekayo.
∎ Umgangatho ophezulu: Amaqhekeza e-RNA acociweyo anobunyulu obuphezulu, awanaprotheyini kunye nobunye ukungcola, kwaye anokumelana nemifuniselo eyahlukahlukeneyo esezantsi komlambo.
Isicelo seKit
Ifanelekile ukutsalwa kunye nokucocwa kwe-RNA iyonke kwiisampuli zezicubu zesityalo esitsha okanye enomkhenkce (ingakumbi izicubu zeqabunga lesityalo esitsha) kunye ne-polysaccharide ephantsi kunye nomxholo we-polyphenol.
Ukuhamba komsebenzi
Umzobo
Isityalo esipheleleyo se-RNA Isolation Kit Plus icutshungulwe i-50mg yamagqabi amatsha e-polysaccharides kunye ne-polyphenols, kunye ne-5% ecocekileyo ye-RNA yavavanywa yi-electrophoresis.
1: Ibhanana
2: Ginkgo
3: Umqhaphu
4: Irharnati
Ukugcinwa kunye nobomi beshelufu
Ikhithi ingagcinwa iinyanga ezili-12 kwiqondo lobushushu begumbi (15–25 ℃) kwindawo eyomileyo, kunye ne-2–8 ℃ ixesha elide (iinyanga ezingama-24).
I-Buffer PSL1 ingagcinwa kwi-4 ℃ kwinyanga ye-1 emva kokongeza i-2-hydroxy-1-ethanethiol (ukhetho).
Isikhokelo sokuHlalutya ingxaki
Olu lucazululo lulandelayo lweengxaki onokudibana nazoIzityalo zizonkeRNA extraction will help you with your experiments. In addition, for other experimental or technical problems in addition to operating instructions and problem analysis, we have dedicated technical support to help you. If you have any needs, please contact us at: 028-83360257 or E-mali : Tech@foregene.com.
Uluhlu olujikelezayo luvalekile
Ukuvinjwa kwekholomu ye-spin kuya kubangela ukuba isivuno se-RNA sincitshiswe okanye singakwazi ukuhlanjululwa ukufumana i-RNA, kwaye umgangatho we-RNA efunyenweyo uya kuba phantsi.
Uhlalutyo lwesizathu esiqhelekileyo:
1. Isampuli ayiphulwanga ngokupheleleyo.
Ukuqhekeka kwesampulu engaphelelanga kunokuthintela i-DNA-Cleaning Column, enokuthi ichaphazele isivuno se-RNA kunye nomgangatho.Sincoma ukuba xa usenza ulwahlulo lwesampulu, ngokukhawuleza ugaye kwinani elaneleyo le-nitrogen engamanzi ukuqhekeza izicubu ezinje ngeendonga zeseli kunye neembrane zeeseli zeesampulu kangangoko kunokwenzeka.Kwiisampuli zezityalo ze-polyphenol polysaccharides, sincoma ukuba usebenzise i-Plant Total RNA Isolation Kit Plus.
2.Aspirate DNA-Cleaning Column yedwa supernatant, aspirate kunokwenzeka cell debris pellet.
I-aspirated cell debris pellet inokuvala iKholamu ye-RNA kuphela ngexesha leenkqubo ze-adsorption ye-RNA (jonga inyathelo lesi-5 lenkqubo, inyathelo lesi-6 lenkqubo ye-polysaccharide polyphenol).Sicebisa ukuba unonophelo kufuneka luthathwe xa unqwenela lo mandla angaphezu kwamandla ukuze uthintele i-debris cell debris.
3. Isixa sokuqala sesampulu sikhulu kakhulu.
Ukusetyenziswa kwesampulu ngokugqithisileyo kuya kubangela ukwahlulwa kwesampulu engaphelelanga okanye uhlaziyo olungaphelelanga lweeseli nge-Buffer PRL1 okanye i-Buffer PSL1, ekhokelela kuluhlu oluvaliweyo lokucoca lwemisebenzi yokucoca.I-Plant Total RNA Isolation Kit inomlinganiselo wokuqala we-50 mg ngokucocwa okukodwa kwesampuli esebenzayo.Kwiisampuli zezityalo ze-polyphenol polysaccharides, sincoma ukuba uzame i-Plant Total RNA Isolation Kit Plus.
4. Ubushushu be-centrifuge buphantsi kakhulu.
Ukwahlulwa okupheleleyo kwe-RNA kunye nokucocwa ngaphandle kokuphazamiseka kwenitrogen ye-liquid yesampula yezicubu, onke amanyathelo enziwa kwiqondo lobushushu legumbi (20-25 °C).Ezinye ii-centrifuge zobushushu obuphantsi zinamaqondo obushushu angaphantsi kwe-20 °C, nto leyo enokubangela imiqobo kwiKholam yokucoca ye-DNA kunye/okanye iKholamu ye-RNA kuphela.Ukuba oku kuyenzeka, seta ubushushu be-centrifuge ukuya kuma-20-25 °C kwaye ufudumeze umxube we-lysis kunye / okanye ukongezwa kwe-ethanol yokwahlula ngaphezulu kwe-37 °C.
Akukho RNA ekhutshiweyo okanye isivuno se-RNA siphantsi
Ngokuqhelekileyo kukho izinto ezininzi ezichaphazela ukusebenza kakuhle kokubuyisela, njenge: umxholo we-RNA yesampuli, indlela yokusebenza, umthamo we-lution, njl.
Uhlalutyo lwezizathu eziqhelekileyo zingezantsi:
I-1.I-ice bath okanye ukushisa okuphantsi (4 ° C) i-centrifugation yenziwa ngexesha lokusebenza.
Isiphakamiso: Sebenza kwiqondo lokushisa (15-25 ° C) kuyo yonke inkqubo, musa ukwenza i-ice bath kunye ne-centrifugation yokushisa ephantsi.
2.I-RNA iye yathotywa ngenxa yokugcinwa okungafanelekanga kwesampuli okanye ukugcinwa kwexesha elide lesampuli.
Isindululo: Iisampulu ezisandula ukuqokelelwa kufuneka zikhe zikhenkcezwe ngokukhawuleza kwinitrogen engamanzi, emva koko zigcinwe ku -80°C ixesha elide, zithintele ukuphinda kukhenkceze kunye nokunyibilika kweisampulu;okanye ngokukhawuleza ucwilise iisampuli kwi-RNA stabilizer RNAlater isisombululo (iisampulu zezilwanyana).
3.Ukwahlulwa kwesampulu eyaneleyo kunye ne-lysis ikhokelela ekuvinjweni kwekholamu yokucoca.
Isiphakamiso: Xa ugaya i-tissue, nceda uqinisekise ukuba i-tissue iphantsi ngokwaneleyo, kwaye ngokukhawuleza uyidlulisele kwi-Buffer ye-PSL1 elungiselelwe ngaphambili (qinisekisa ukuba umlinganiselo ochanekileyo we-β-ME wongezwe, jonga inyathelo 1 lenkqubo).
4.I-eluent yongezwa ngokungalunganga.
Ingcebiso: Qinisekisa ukuba iRNase-Free ddH2I-O ithontsizwa phakathi kwe-membrane yekholomu yokucoca.
5.Umthamo ochanekileyo we-ethanol epheleleyo ayizange yongezwe kwi-Buffer PSL2 okanye i-Buffer PRW2.
Ingcebiso: Nceda ulandele imiyalelo, yongeza umthamo ochanekileyo we-ethanol epheleleyo kwi-Buffer PSL2 kunye ne-Buffer PRW2 kwaye udibanise kakuhle phambi kokuba ikhithi isetyenziswe.
6.Ubungakanani besampulu yethishu ayifanelekanga.
Ingcebiso: Sebenzisa i-50 mg yethishu nge-500 μl ye-Buffer PSL1.Ukusebenzisa izicubu ezininzi kakhulu kuya kunciphisa inani le-RNA ekhutshiweyo kwaye ukucoceka kwe-RNA okubangelwayo kuya kuncitshiswa.Sincoma kakhulu ukuba idosi yesampulu yokuqala ayifanele idlule i-50 mg ngomsebenzi wokutsalwa kwe-RNA.
7. Umthamo wesandi esingafanelekanga okanye i-elution engaphelelanga.
Isiphakamiso: Umthamo ocacileyo wekholamu yokucoca ngu-50-200 μl;ukuba isiphumo se-elution asanelisi, kuyacetyiswa ukuba kwandiswe ixesha kwiqondo lobushushu legumbi emva kokongeza i-RNase-Free ddH efudunyeziweyo.2O, njenge-5-10min.
8.Ikholamu yokucoca inentsalela ye-ethanol emva kokuhlamba nge-Buffer PRW2.
Isiphakamiso: Ukuba ityhubhu engenanto i-centrifuged ye-1 min kwaye kusekho i-ethanol eseleyo emva kokuhlamba kwi-Buffer PRW2, unokwandisa ixesha le-tube engenanto ye-centrifugation ukuya kwi-2 min, okanye ubeke ikholamu yokucoca kwindawo yokushisa ye-5 min ukususa ngokupheleleyo i-ethanol eseleyo.
9.Ikhithi isetyenziswe ngokungachanekanga.
Isiphakamiso: Kwiisampuli zezityalo ze-polyphenolic polysaccharides, ukusebenzisa iikiti eziqhelekileyo ezifana ne-Plant Total RNA Isolation Kit ayinakukwazi ukufumana iisampulu ezifanelekileyo ze-RNA.Sincoma ukuba usebenzise iPlant Total RNA IsolationKit Plus, eyilelwe ngokukodwa iisampuli zezityalo ze-polyphenolic polysaccharide.Ikhithi ephuhliswe ngokukodwa ukukhupha i-RNA kwi-polyphenol kunye neesampuli zezityalo ze-polysaccharide.
Ixabiso le-OD260/OD280 liphantsi
I-RNA elution kunye ne-ddH2I-O kwaye isetyenziselwa ukufundwa kwe-spectrophotometer iphumela kwixabiso eliphantsi le-OD260/OD280.Sincoma ukusebenzisa i-10 mM Tris-HCl, pH 7.5 (kunokuba RNase-Free ddH2O to elute RNA) ukufumana amaxabiso achanekileyo eOD260/OD280, bona “RNA Concentration and Purification Assays” kwiphepha le-19.
I-RNA ehlanjululweyo ithotywa
Umgangatho we-RNA ecocekileyo inxulumene nezinto ezifana nokugcinwa kwesampulu, ukungcoliseka kwe-RNase, kunye nokukhwabanisa.
Uhlalutyo lwezizathu eziqhelekileyo:
I-1.Iisampulu ze-tissue azizange zigcinwe ngexesha emva kokuqokelela.
Isincomo: Ukuba iisampulu zezicubu azisetyenziswanga ngexesha emva kokuqokelela, nceda uzigcine kwi-nitrogen engamanzi kwiqondo lokushisa eliphantsi ngokukhawuleza okanye uzidlulisele kwi--80 ° C yokugcina ixesha elide emva kokuqhwala ngokukhawuleza kwi-nitrogen engamanzi, okanye ngokukhawuleza ucwilise iisampuli kwi-RNA stabilizer RNAlater isisombululo (iisampulu zezilwanyana).Ukukhutshwa kwe-RNA, zama ukusebenzisa iisampulu zeethishu ezisanda kuqokelelwa.
2.Ukukhenkceza okuphindaphindiweyo kunye nokunyibilika kweesampuli zethishu.
Icebiso: Xa ugcina iisampulu zethishu, kungcono ukuzisika zibe ziingceba ezincinci ukuze zigcinwe, kwaye ukhuphe inxalenye yazo xa uzisebenzisa ukuthintela ukonakaliswa kweRNA okubangelwa kukukhenkcezwa okuphindaphindiweyo nokunyibilika kweisampulu.
3.I-RNase yaziswa kwigumbi lokusebenza okanye inganxitywanga iiglavu ezilahlwayo, iimaski, njl.
Ingcebiso: Imifuniselo yokutsalwa kwe-RNA yenziwa ngcono kwimisebenzi eyahlukeneyo ye-RNA, kwaye itafile yelabhoratri kufuneka icocwe phambi kovavanyo, kwaye iiglavu ezilahlwayo kunye nemaski kufuneka zinxitywe ngexesha lovavanyo ukunqanda ukuthotywa kwe-RNA okubangelwa kukwaziswa kwe-RNase ngowona mgangatho mkhulu.
I-4.I-reagent ingcolisekile yi-RNase ngexesha lokusetyenziswa.
Ingcebiso: Faka endaweni yoluhlu olutsha lwezixhobo ze-RNA zokutsalwa kwezityalo zizonke zemifuniselo enxulumeneyo.
I-5.Iibhubhu ze-centrifuge kunye neengcebiso ze-pipette ezisetyenziselwa ukuguqulwa kwe-RNA zingcolile nge-RNase.
Ingcebiso: Qinisekisa ukuba iityhubhu ze-centrifuge, iingcebiso ze-pipette, ii-pipettes, njl njl. ezisetyenziswa kwi-RNA extraction zonke zi-RNase-Free.
Izikhokelo zoMyalelo:
