ISeli iyonke yeRNA Isolation Kit Iyonke iRNA Isolation IiKits zokucoca ukusuka kwiseli
Inkcazelo yeKit:
Isulungekiswe kakhulu kwaye ikumgangatho ophezulu we-RNA iyonke inokufumaneka kwiiseli ezahlukeneyo zenkcubeko kwimizuzu eli-11.
RNase-Free
Isebenza kubushushu begumbi (15-25℃)
Nge-DNA-Ukucoca iKholam
Isivuno esiphezulu se-RNA
Ukukhawuleza:Gqiba ukutsalwa ngemizuzu eyi-11
Ukhuseleko:Ayikho imichiza ye-Organic
Inkcazo
Le kit isebenzisa ikholomu ye-spin kunye nefomula ephuhliswe yi-Foregene, enokuthi ikhuphe ngokufanelekileyo ubunyulu obuphezulu kunye nomgangatho ophezulu we-RNA kwiiseli ezikhuliswe kwi-96, i-24, i-12, kunye ne-6-well-plates.
Ikiti ibonelela ngeKholamu ye-DNA-Cleaning esebenzayo, ekwazi ukwahlula ngokulula i-supernatant kunye ne-cell lysate, ibophe kwaye isuse i-genomic DNA.Umsebenzi ulula kwaye wonga ixesha.
Tyena iKholamu ye-RNA kuphela inokubopha ngokufanelekileyo i-RNA ngefomula eyodwa.Inani elikhulu leisampulu linokucutshungulwa ngaxeshanye.
Amacandelo ekhithi
| Ukwakhiwa kweKit | RE-03111 | RE-03114 |
| 50 T | 200 T | |
| Isithinteli cRL1* | 25 ml | 100 ml |
| Isithinteli cRL2 | 15 ml | 60 ml |
| Isithinteli RW1* | 25 ml | 100 ml |
| Isithinteli RW2 | 24 ml | 96 ml |
| RNase-Free ddH2O | 10 ml | 40 ml |
| Ikholamu ye-RNA Kuphela | 50 | 200 |
| Ikholamu yokucoca iDNA | 50 | 200 |
| Umyalelo | 1 | 1 |
*Nceda unxibe iiglavu kwaye uthathe amanyathelo okukhusela ngexesha lomsebenzi njengoko i-Buffer cRL1 kunye ne-Buffer RW1 ziqulethe iityuwa ze-chaotropic ezicaphukisayo.
Iimpawu&izinto eziluncedo
■ Yonke le nkqubo iqhutywa kubushushu begumbi (15-25℃), ngaphandle kokuhlamba komkhenkce kunye nobushushu obuphantsi be-centrifugation.
■ Ikhithi iyonke i-RNase-Free, akukho mfuneko yakuzikhathaza ngokonakala kwe-RNA.
∎ IKholam yokucoca i-DNA ibophelela ngokukodwa i-DNA, ukuze ikhithi ikwazi ukususa ungcoliseko lwe-DNA ye-genomic 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 ngemizuzu eyi-11.
■ Ukhuseleko: Akukho sixhobo sokwenza izinto eziphilayo esifunekayo.
∎ Umgangatho ophezulu: I-RNA ecociweyo yeyobunyulu obuphezulu, ayinaprotheyini kunye nobunye ukungcola, kwaye inokumelana nemifuniselo eyahlukeneyo elandelayo.
Isicelo seKit
Ifanelekile ukutsalwa kunye nokuhlanjululwa kwe-RNA epheleleyo kwiiseli ezikhuliswe kwii-96, 24, 12, kunye ne-6-well-plates.
Ukuhamba komsebenzi
Umzobo
Umzobo webhetri yejeli ye-agarose yeCell Total RNA Isolation Kit yaphatha amanani angasentla ahlukeneyo eeseli, 20μl umthamo we-elution, thatha i-2μl esulungekisiweyo iyonke ye-RNA 1%.
Ukugcinwa kunye nobomi beshelufu
Ikhithi ingagcinwa iinyanga ezili-12 kwiqondo lobushushu begumbi (15–25 ℃) okanye 2–8 ℃ ixesha elide (iinyanga ezingama-24).
I-Buffer cRL1 inokugcinwa kwi-4 ℃ kwinyanga ye-1 emva kokongeza i-2-hydroxy-1-ethanethiol (ukhetho).
I-RNA ayikhutshwa okanye izivuno ze-RNA ziphantsi
Kuhlala kukho izinto ezahlukeneyo ezichaphazela ukusebenza kakuhle kokubuyisela, njengale: umxholo we-RNA we-tissue, indlela yokusebenza, umthamo we-lution, njl.
1. Ibhafu ye-ice okanye i-cryogenic (4 ° C) i-centrifugation yenziwa ngexesha lokusebenza.
Isincomo: Sebenza kwiqondo lokushisa (15-25 ° C) kuyo yonke inkqubo, musa ukuhlamba i-ice kunye ne-centrifuge kumaqondo aphantsi.
2. Ukugcinwa kwesampulu engafanelekanga okanye ixesha lokugcina isampuli.
Isindululo: Gcina iisampulu kwi -80 °C okanye umkhenkce kwinitrogen engamanzi kwaye uthintele ukusetyenziswa okuphindaphindiweyo komkhenkce;zama ukusebenzisa izicubu ezitsha okanye iiseli ezikhuliswayo zokutsalwa kwe-RNA.
3. I-lysis yesampuli eyaneleyo.
Isincomo: Xa i-homogenizing tissue, qinisekisa ukuba i-tissue i-homogenized ngokwaneleyo kwaye iiseli zethishu zahlulwe ngokwaneleyo ukucacisa ukukhululwa kwe-RNA.
4. I-eluent ayidityaniswa ngokuchanekileyo.
Ingcebiso: Qinisekisa ukuba i-RNase-Free ddH2I-O yongezwa kwi-dropwise phakathi kwe-membrane yekholomu yokucoca.
5. Umthamo ochanekileyo we-absolute ethanol awuzange wongezwe kwi-Buffer RL2 okanye kwi-Buffer RW2.
Ingcebiso: Landela imiyalelo, yongeza umthamo ochanekileyo we-ethanol epheleleyo kwi-Buffer RL2 kunye ne-Buffer RW2 kwaye udibanise kakuhle phambi kokusebenzisa ikhithi.
6. Idosi yesampula yezicubu ayifanelekanga.
Isincomo: Sebenzisa i-10-20 mg yezicubu okanye (1-5) × 106iiseli nge-500 μl buffer RL1, njengoko ukusetyenziswa kwethishu ngokugqithisileyo kunokubangela ukutsalwa kwe-RNA okucuthiweyo.
7. Umthamo ongafanelekanga okanye i-elution engaphelelanga.
Isincomo: Umthamo we-elution wekholamu yokucoca yi-50-200 μl;ukuba isiphumo se-elution asanelisi, kuyacetyiswa ukuba kwandiswe ixesha lokubeka ubushushu begumbi emva kokongeza i-RNase-Free ddH efudunyeziweyo.2O, umz. kwi-5-10 min.
8.Ikholamu yokucoca inentsalela ye-ethanol emva kokuhlamba kwe-Buffer RW2.
Isincomo: Ukuba kukho intsalela ye-ethanol emva kokuhlamba kwe-Buffer RW2, i-tube engenanto ye-centrifugation ye-1min, ixesha le-tube engenanto yokusebenza kwe-centrifugation linganyuswa ukuya kwi-2min, okanye ikholomu yokucoca inokubekwa kwindawo yokushisa ye-5 min ukususa ngokwaneleyo i-ethanol eseleyo.
I-RNA ecocekileyo ithotywa
Umgangatho we-RNA ecocekileyo inxulumene nezinto ezifana nokugcinwa kwesampulu, ukungcoliswa kwe-RNase, kunye nokukhwabanisa, njl.
1. Iisampulu zezicubu azigcinwanga ngexesha.
Isincomo: Ukuba iisampulu zethishu okanye iiseli azisetyenziswanga ngexesha elifanelekileyo emva kokuqokelelwa, ngoko nangoko gcina i-cryopreserve kwi -80 °C okanye initrogen engamanzi.Ukukhupha i-RNA, sebenzisa ithishu esanda kuthathwa okanye isampulu yeseli xa kunokwenzeka.
2. Ukunyibilika okuphindaphindiweyo kweesampuli zethishu.
Isincomo: Xa ugcina iisampulu zethishu, kungcono ukuba uzinqumle zibe ngamaqhekeza amancinci ukuze zigcinwe, kwaye ususe esinye seziqwenga xa uzisebenzisa ukuphepha ukunyibilika okuphindaphindiweyo kwesampulu kunye nokuthotywa kwe-RNA.
3. I-RNase yaziswa okanye ayinxibi iiglavu ezilahlwayo, iimaski, njl njl ngexesha lokusebenza.
Isincomo: Imifuniselo yokutsalwa kwe-RNA yenziwa ngcono kumagumbi okukhohlisa e-RNA kwaye itafile iyacinywa phambi kovavanyo.
Nxiba iiglavu ezilahlwayo kunye nemaski ngexesha lovavanyo lokunciphisa ukuthotywa kwe-RNA okubangelwa kukwaziswa kwe-RNase.
4. Ii-reagents zingcolisekile nge-RNase ngexesha lokusetyenziswa.
Ingcebiso: Faka endaweni entsha yeSilwanyana esipheleleyo se-RNA ye-Isolation Kit ukulungiselela imifuniselo ehambelanayo.
5. Iityhubhu ze-centrifuge, iingcebiso, njl.njl ezisetyenziswa kwi-RNA manipulation zingcolile nge-RNase.
Ingcebiso: Qinisekisa ukuba iityhubhu ze-centrifuge, iingcebiso, iipipette, njl.njl ezisetyenziswa kwi-RNA extraction azinayo yonke i-RNase.
I-RNA esulungekileyo ichaphazela imifuniselo esezantsi
I-RNA ihlanjululwe yikholomu yokucoca, ukuba iiyoni zetyuwa, umxholo weprotheyini mkhulu kakhulu uya kuchaphazela umfuniselo osezantsi, onje: uguqulelo lombhalo, Northern Blot et al.
1. I-RNA ekhutshiweyo ineentsalela ze-ion yetyuwa.
Isincomo: Qinisekisa ukuba umthamo ochanekileyo we-ethanol wongezwe kwi-Buffer RW2 kwaye wenze i-2 ikholamu yokuhlanjululwa kwi-speed ye-centrifugal eboniswe ekusebenzeni;ukuba kukho nayiphi na intsalela ye-ion yetyuwa, shiya ikholomu yokucoca kwi-Buffer RW2 ye-5 min kwiqondo lokushisa elingaphakathi kwaye wenze i-centrifugation ukwandisa ukukhutshwa kwetyuwa.
2. Intsalela ye-Ethanol kwi-RNA elutioned.
Isincomo: Qinisekisa ukuba emva kokuhlamba i-buffer RW2, yenza i-tube engenanto ye-centrifugation operation kwisantya se-centrifugation esibonakaliswe ukusebenza, ukwandisa ixesha le-tube engenanto ye-centrifugation operation ukuya kwi-2 min ukuba kusekho i-ethanol intsalela, okanye uyishiye kwindawo yokushisa kwe-5 m.
