I-Plant Total RNA Isolation Kit Plus Isamba se-RNA Purificaiton Kit Yezitshalo Ezicebile kuma-Polysaccharides nama-Polyphenols
Incazelo yekhithi:
I-Cat.No.RE-05021/05022/05024
Ukuze kuhlanzwe isamba se-RNA kumasampula ezitshalo ezivamile aqukethe i-polysaccharide ephezulu nezingxenye ze-polyphenol.
Khipha ngokushesha isamba sekhwalithi ephezulu ye-RNA kumasampula ezitshalo anokuqukethwe okuphezulu kwama-polysaccharides nama-polyphenols.
I-RNase-Free Ukusebenzisa Ikholomu Yokuhlanza I-DNA
Kulula—yonke imisebenzi iqedwa ezingeni lokushisa legumbi
Ukusheshisa—ukusebenza kungaqedwa ngemizuzu engu-30
Iphephile—ayikho i-organic reagent esetshenzisiwe 
Imininingwane
50 Preps, 200 Preps
Ikhithi isebenzisa ikholomu yokuphotha kanye nefomula eyakhiwe yi-Foregene, engakhipha ngempumelelo i-RNA ephezulu nekhwalithi ephezulu ezicutshini zezitshalo ezihlukahlukene ezinokuqukethwe okuphezulu kwama-polysaccharides noma ama-polyphenols.Ihlinzeka ngekholomu ye-DNA-Cleaning engasusa kalula i-genomic DNA ku-supernatant kanye ne-tissue lysate.Ikholomu ye-RNA kuphela ingabopha ngempumelelo i-RNA.Ikhithi ingacubungula inani elikhulu lamasampuli ngesikhathi esisodwa.
Lonke uhlelo aluqukethe i-RNase, ngakho-ke i-RNA ehlanziwe ngeke yehliswe.I-Buffer PRW1 kanye ne-Buffer PRW2 ingaqinisekisa ukuthi i-RNA etholiwe ayingcoliswanga amaprotheni, i-DNA, ama-ion, nezinhlanganisela eziphilayo.
Izingxenye zekhithi
| Buffer PSL1, Buffer PS, Buffer PSL2 |
| Isilondolozi PRW1, Isivimbeli PRW2 |
| I-ddH yamahhala ye-RNase2O, Ikholomu Yokuhlanza I-DNA |
| Ikholomu Ye-RNA Kuphela |
Izici&izinzuzo
■ Ukusebenza ekamelweni lokushisa (15-25℃) kuyo yonke inqubo, ngaphandle kokugeza eqhweni nokushisa okuphansi kwe-centrifugation.
■ Ikhithi ephelele ye-RNase-Free, asikho isidingo sokukhathazeka ngokuwohloka kwe-RNA.
■ Ifaneleka ngokukhethekile ukuhlanzwa kwe-RNA kumasampula ezitshalo ama-polysaccharides nama-polyphenols.
■ Ikholomu Yokuhlanza I-DNA ibophezela ngokukhethekile ku-DNA, ukuze ikhithi isuse ukungcoliswa kwe-DNA ye-genomic ngaphandle kokwengeza i-DNase.
■ Isivuno esikhulu se-RNA: Ikholomu ye-RNA kuphela kanye nefomula eyingqayizivele ingahlanza i-RNA ngokuphumelelayo.
■ Isivinini esisheshayo: kulula ukusebenza futhi singaqedwa phakathi nemizuzu engama-30.
■ Ukuphepha: asikho i-reagent ephilayo edingekayo.
■ Ikhwalithi ephezulu: Izingcezwana ze-RNA ezihlanziwe zimsulwa kakhulu, azinawo amaphrotheni nokunye ukungcola, futhi zingahlangabezana nezinhlelo zokuhlola ezihlukahlukene ezansi nomfula.
Imingcele yomkhiqizo
■ Izinhlelo zokusebenza eziwela phansi: ukuhlanganiswa kwe-cDNA ye-first-strand, i-RT-PCR, i-molecular cloning, i-Northern Blot, njll.
■ Isampula: Izicubu zezitshalo ezintsha noma eziqandisiwe zama-polysaccharides nama-polyphenols
■ Umthamo: 50mg izicubu zezitshalo
■ Umthamo omkhulu wokubopha we-RNA wekholomu yokuhlanza: 80 μg
■ Ivolumu ye-Elution: 50-200 μl
Isicelo sekhithi
Ilungele ukukhishwa nokuhlanzwa kwe-RNA ephelele kumasampula ezicubu zezitshalo ezintsha noma eziqandisiwe (ikakhulukazi izicubu zamaqabunga ezitshalo ezintsha) ezinokuqukethwe okuphezulu kwe-polysaccharide kanye ne-polyphenol.
Ukugeleza komsebenzi
Umdwebo
I-Plant Total RNA Isolation Kit Plus icutshungulwe u-50mg wamaqabunga amasha e-polysaccharides nama-polyphenols, futhi i-RNA ehlanzekile engu-5% yahlolwa nge-electrophoresis.
1: Ubhanana
2: Ginkgo
3: Ukotini
4: Ihalanathi
Isitoreji kanye Nempilo yeshelufu
Le kit ingagcinwa izinyanga ezingu-24 ngaphansi kwezimo ezomile ekamelweni lokushisa (15-25℃);uma idinga ukugcinwa isikhathi eside, ingagcinwa ku-2–8℃.
I-Buffer PSL1 ingafakwa kokuthi 4℃ inyanga engu-1 ngemva kokwengeza i-β-mercaptoethanol (kuyanconywa ukuthi iyengeze ngesikhathi esifanayo sokuhlola).
Ikholomu ixhunyiwe
Ngemuva kokuthi ikholomu ixhunyiwe, isivuno se-RNA siyancishiswa noma akunakwenzeka ukuhlanza i-RNA, futhi isisindo esitholiwe se-RNA siphansi.
Ukuhlaziywa kwembangela evamile:
1. Ukuphumula kwesampula akuphelele.
Ukuphuka kwesampula akubangeli ngokuphelele i-DNA-CLEANING COLUMN ukuthi ivinjwe, kuyilapho kuthinta isivuno nekhwalithi ye-RNA.Sincoma ukusebenza kokugaya ngokushesha ku-nitrogen ewuketshezi eyanele lapho uphula amasampula, Zama ukuchoboza udonga lwamaseli wesampula, ulwelwesi lwamaseli nezinye izicubu.Kumasampula ezitshalo e-polysaccharides, sincoma ukuthi usebenzise I-Plant Total RNA IsolATION KIT PLUS.
2. Uma umunca isampula ye-supernatant ehlukanisiwe Ngekholomu Yokuhlanza Ye-DNA, i-precipitate yeseli engaba yizicucu ingase ihogelwe.
Izinsalela ezihlukene zeseli ezithathiwe zizobangela Ikholomu YE-RNA KUPHELA ezovinjwa lapho kwenziwa umsebenzi wokukhangisa we-RNA (bona isinyathelo 6).Sincoma ukuthi uqaphele lapho uncela lesi sidakamizwa esinamandla ukuze ugweme ukumuncwa ama-cell debris.
3. Inani lokuqala lesampula likhulu kakhulu.
Ukusetshenziswa kwesampula okweqile kuzoholela ekuhlukaneni kwesampula okungaphelele noma ukuguqulwa kweseli okungaphelele nge-Buffer PSL1, okuholela ekuvinjweni kwekholomu yokuhlanza ngesikhathi sokuhlanza.Ikhithi Yokuhlukanisa Yezitshalo Isamba se-RNA Isampula ngayinye yokusebenza ehlanziwe ingu-50 mg.Ngamasampula ezitshalo e-polysaccharides, sincoma ukuthi uzame I-Plant Total RNA IsolATION KIT PLUS.
4. Izinga lokushisa le-centrifuge liphansi kakhulu.
Yonke inqubo yokuhlukaniswa kwe-RNA kanye nenqubo yokuhlanza yenziwa ekamelweni lokushisa (20-25°C), ngaphandle kokuthi isicubu sesampula siphulwa yi-nitrogen ewuketshezi.Izinga lokushisa kwamanye ama-cryogenic centrifuges lingaphansi kuka-20℃, okungase kubangele ukuvinjwa Kwekholomu Yokuhlanza I-DNA kanye/noma Ikholomu Ye-RNA Kuphela.Uma lokhu kwenzeka, setha izinga lokushisa le-centrifuge libe ngu-20-25℃, futhiqiniseka ukuthi ingxube ye-lysis kanye/noma i-ethanol-added supernatant ishiselwe kuze kube ngu-37°C.
Ayikho i-RNA ekhishiwe noma isivuno se-RNA siphansi
Ngokuvamile kuba nezinto eziningi ezithinta ukusebenza kahle kokuthola kabusha, njengalezi: isampula yokuqukethwe kwe-RNA, indlela yokusebenza, ivolumu ye-elution, njll.
Ukuhlaziywa kwezimbangela ezivamile njengoba ngezansi:
I-1.Ukugeza kweqhwa noma izinga lokushisa eliphansi (4 ° C) i-centrifugation yenziwa ngesikhathi sokusebenza.
Ukusikisela: Isebenza ekamelweni lokushisa (15-25°C) kuyo yonke inqubo, ungenzi ukugeza kweqhwa kanye ne-centrifugation yokushisa ephansi.
2.I-RNA yehlisiwe ngenxa yokulondolozwa okungafanele kwesampula noma ukulondolozwa kwesikhathi eside kwesampula.
Okutuswayo: Amasampula asanda kuqoqwa kufanele aqandwe ngokushesha ku-nitrogen ewuketshezi, abese egcinwa ku -80°C isikhathi eside, agweme ukuqhwaza okuphindaphindiwe nokuncibilika kwamasampuli;noma ngokushesha cwilisa amasampula ku-RNA stabilizer RNAlater solution (amasampula ezilwane).
3.Ukuhlukaniswa kwesampula okunganele kanye ne-lysis kuholela ekuvinjweni kwekholomu yokuhlanza.
Isiphakamiso: Uma ugaya izicubu, sicela uqinisekise ukuthi isicubu sigaywe ngokwanele, futhi usidlulisele ngokushesha ku-Buffer PSL1 esilungiselelwe ngaphambilini (qinisekisa ukuthi ingxenye elungile ye-β-ME yengeziwe, bona isinyathelo 1 senqubo).
4.I-eluent yengezwe ngokungalungile.
Isiphakamiso: Qiniseka ukuthi i-ddH2O ye-RNase-Free iconsiselwa phakathi nolwelwesi lwekholomu yokuhlanza.
5.Ivolumu elungile ye-ethanol ephelele ayizange yengezwe ku-Buffer PSL2 noma ku-Buffer PRW2.
Ukusikisela: Sicela ulandele imiyalelo, wengeze ivolumu efanele ye-ethanol ephelele ku-Buffer PSL2 kanye ne-Buffer PRW2 bese uxuba kahle ngaphambi kokuba ikhithi isetshenziswe.
6.Inani lesampula lethishu alifanelekile.
Isiphakamiso: Sebenzisa u-50 mg wethishu ngo-500 μl we-Buffer PSL1.Ukusebenzisa izicubu eziningi kuzonciphisa inani le-RNA ekhishiwe futhi ukuhlanzeka kwe-RNA okuwumphumela nakho kuzoncipha.Sincoma ngokuqinile ukuthi umthamo wokuqala wesampula akufanele udlule u-50 mg ngokusebenza kokukhipha kwe-RNA ngakunye.
7.Ivolumu ye-elution engafanele noma ukungezwani okuphelele.
Isiphakamiso: Umthamo ongacacile wekholomu yokuhlanza ungama-50-200 μl;uma umphumela we-elution ungagculisi, kuyanconywa ukuthi unwebe isikhathi kuzinga lokushisa legumbi ngemva kokwengeza i-ddH2O eshisiwe ye-RNase-Free, njenge-5-10min.
8.Ikholomu yokuhlanza inensalela ye-ethanol ngemva kokugeza nge-BufferPRW2.
Isiphakamiso: Uma ishubhu elingenalutho li-centrifuged iminithi elingu-1 futhi kusekhona i-ethanol esele ngemva kokugeza ku-Buffer PRW2, ungakwazi ukwandisa isikhathi se-tube centrifugation engenalutho sibe yimizuzu emi-2, noma ubeke ikholomu yokuhlanza ekamelweni lokushisa imizuzu emi-5 ukuze ususe ngokuphelele i-ethanol esele.
9.Ikhithi isetshenziswe ngokungalungile.
Isiphakamiso: Kumasampula ezitshalo we-polyphenolic polysaccharides, ukusebenzisa amakhithi avamile njenge-Plant Total RNA Isolation Kit kungase kungakwazi ukuthola amasampula e-RNA afanelekile.Sincoma ukuthi usebenzise i-Plant Total RNA IsolationKit Plus, eklanyelwe ngokukhethekile amasampula ezitshalo ze-polyphenolic polysaccharide.Ikhithi eyakhelwe ngokukhethekile ukukhipha i-RNA ku-polyphenol namasampula ezitshalo ze-polysaccharide.
OD260/OD280 inani liphansi
I-RNA elution ene-ddH2O futhi isetshenziselwa ukufundwa kwe-spectrophotometer iphumela kumanani aphansi e-OD260/OD280.Sincoma ukusebenzisa u-10 mM Tris-HCl, pH 7.5 (kunokuba i-RNase-Free ddH2O ukuze ukhiphe i-RNA) ukuze uthole amanani alungile uma kuqhathaniswa e-OD260/OD280, bona “I-RNA Concentration and Purification Assays” ekhasini 19.
I-RNA ehlanziwe yehlisiwe
Ikhwalithi ye-RNA ehlanzekile ihlobene nezici ezifana nokulondolozwa kwesampula, ukungcoliswa kwe-RNase, nokukhohlisa.
Ukuhlaziywa kwezimbangela ezijwayelekile:
1.Amasampula ezicubu awazange agcinwe ngesikhathi ngemuva kokuqoqwa.
Okutuswayo: Uma amasampula ezicubu engasetshenziswa ngesikhathi ngemva kokuqoqwa, sicela uwagcine ku-nitrogen ewuketshezi ezingeni lokushisa eliphansi ngokushesha noma uwadlulisele ku--80°C ukuze agcinwe isikhathi eside ngemva kokuqhwaza okusheshayo ku-nitrogen ewuketshezi, noma ngokushesha ucwilise amasampula ku-RNA stabilizer RNAlater solution (amasampula ezilwane ).Ukuze uthole i-RNA, zama ukusebenzisa amasampula ezicubu ezisanda kuqoqwa.
2.Ukuqhwaza okuphindaphindiwe nokuncibilika kwamasampula ezicubu.
Ukusikisela: Lapho ugcina amasampula ezicubu, kungcono ukuwasika abe izingcezu ezincane ukuze alondolozwe, futhi ukhiphe ingxenye yawo lapho uwasebenzisa ukuze ugweme ukuwohloka kwe-RNA okubangelwa ukubanda okuphindaphindiwe nokuncibilika kwamasampula.
3.I-RNase yethulwa egunjini lokuhlinza noma engagqoki amagilavu alahlwayo, imaski, njll.
Isiphakamiso: Ukuhlolwa kokukhipha i-RNA kwenziwa kangcono kakhulu emisebenzini ehlukene ye-RNA, futhi ithebula laselabhorethri kufanele lihlanzwe ngaphambi kokuhlolwa, futhi amagilavu alahlwayo namamaski kufanele kugqokwe phakathi nokuhlolwa ukuze kugwenywe ukuwohloka kwe-RNA okubangelwa ukwethulwa kwe-RNase ngezinga elikhulu kakhulu.
4.I-reagent ingcoliswe yi-RNase ngesikhathi sokusetshenziswa.
Isiphakamiso: Faka esikhundleni ngochungechunge olusha lwamakhithi okukhipha i-RNA esitshalo esiphelele sokuhlolwa okuhlobene.
5.Amashubhu e-centrifuge namathiphu we-pipette asetshenziselwa ukukhwabanisa kwe-RNA angcoliswe yi-RNase.
Ukusikisela: Qinisekisa ukuthi amashubhu e-centrifuge, amathiphu e-pipette, ama-pipette, njll. asetshenziswa ekukhishweni kwe-RNA konke Akuna-RNAse.
Amamanuwali wemiyalo:
I-Plant Total RNA Isolation Kit Plus Instruction Manual
