RNAprep Kwakachena Cell / Bhakitiriya Kit
Zvimiro
Buff Zvigadziriso zvakagadziridzwa zvemasero akakwenenzverwa nema sampu ebhakitiriya zvinoita kuti maitiro acho ave nyore uye nyore.
Uni Yakasarudzika DNase I inoderedza genomic DNA kusvibiswa.
■ Yakasarudzika RNase-Yemahara Kuchenesa Makoramu CS inobvisa kumwe kusvibiswa.
■ Iyo yakachena-kuchena uye yakagadzirira-kushandisa RNA inokodzera kune inoyerera yekudzika mafomu.
■ Hapana phenol / chloroform yekudzinga, hapana LiCl uye ethanol kunaya, hapana CsCl gradient centrifugation inodiwa, izvo zvinoita kuti maitiro acho ave akachengeteka uye akavimbika.
Zvishandiso
■ RT-PCR.
■ Northern Blot, Dot Blot.
■ Real-Nguva PCR.
■ Chip kuongorora.
■ PolyA Kuongorora, in vitro dudziro, RNase yekudzivirira ongororo uye mamorekuru kuumbana.
Zvese zvigadzirwa zvinogona kuve zvakagadziriswa ODM / OEM. Kuti uwane mamwe mashoko,ndokumbira ubaye Customized Service (ODM / OEM)
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Zvinyorwa: Evanhu Jurkat Maseru (1 × 106 ) Maitiro: Iyo yakazara RNA yeVanhu Jukat Maseru yaive yakasarudzika ichishandisa iyo RNAprep Yakachena Cell / Bhakitiriya Kit. Mhedzisiro: Ndokumbira kuti uone iri pamusoro agarose gel electrophoresis pikicha. 2-4 μl ye50 μl eluates akatakurwa pamugwagwa. Iyo electrophoresis yakaitwa pa6 V / cm kwemaminitsi makumi matatu pa1% agarose gel. |
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Zvinyorwa: TOP10 E.coli (1 × 108) Maitiro: Iyo yakazara RNA yeTOP10 E.coli yaive yakasarudzika ichishandisa iyo RNAprep Kwakachena Cell / Bhakitiriya Kit. Mhedzisiro: Ndokumbira kuti uone iri pamusoro agarose gel electrophoresis pikicha. 2-4 μl ye50 μl eluates akatakurwa pamugwagwa. Iyo electrophoresis yakaitwa pa6 V / cm kwemaminitsi makumi matatu pa1% agarose gel. |
A-1 Cell lysis kana homogenization haina kukwana
- Deredza kushandiswa kwesampula, wedzera huwandu hwerudzi rwe lysis, wedzera homogenization uye lysis nguva.
A-2 Mhando yemuenzaniso yakanyanya kukura
--Deredza huwandu hwemuenzaniso wakashandiswa kana kuwedzera huwandu hwerisiti bhaisikopo.
A-1 Isina kukwana sero lysis kana homogenization
- Deredza kushandiswa kwesampula, wedzera huwandu hwerudzi rwe lysis, wedzera homogenization uye lysis nguva.
A-2 Mhando yemuenzaniso yakanyanya kukura
- Ndokumbira utarise kunonyanyisa kugadzira chinzvimbo.
A-3 RNA haina kukwidziridzwa zvizere kubva pambiru
- Mushure mekuwedzera RNase-Yemahara mvura, siya iyo kwemaminetsi mashoma pamberi pe centrifuging.
A-4 Ethanol iri mudenga
- Mushure mekuchenesa, centrifuge zvakare uye bvisa iyo yekugezera bhafa zvakanyanya sezvinobvira.
A-5 Cell tsika yepakati haina kubviswa zvachose
- Paunenge uchiunganidza maseru, ndapota ita chokwadi chekubvisa iyo tsika yepakati zvakanyanya sezvinobvira.
A-6 Masero akachengetwa muRNAstore haana kunyatsoita centrifuged
- RNAstore density yakakura kupfuura avhareji sero tsika svikiro; saka simba re centrifugal rinofanira kuwedzerwa. Inokurudzirwa kucentrifuge pa3000x g.
A-7 Yakadzika RNA zvemukati uye kuwanda mune muyenzaniso
- Shandisa yakanaka sampuro kuona kana yakaderera-goho inokonzerwa nesampuli.
A-1 Chinyorwa hachisi chitsva
- Nyowani nyowani inofanirwa kuchengetwa muiriidhi senitrogen nekukasira kana nekukurumidza kuiswa muRNAstore reagent kuve nechokwadi chekudzora mhedzisiro.
A-2 Mhando yemuenzaniso yakanyanya kukura
- Deredza sampuro yemari.
A-3 RNase kusvibiswan
- Kunyangwe iyo buffer yakapihwa mune kit haina RNase, zviri nyore kusvibisa RNase panguva yekubvisa uye inofanira kubatwa nehanya.
A-4 Electrophoresis kusvibiswa
- Tsiva iyo electrophoresis buffer uye ita shuwa kuti zvinoshandiswa uye Kurodha Buffer hazvina kusvibiswa neRNase.
A-5 Kunyanya kurodha electrophoresis
--Deredza huwandu hwesampuli kurodha, kurodha kwetsime rega rega hakufanirwe kupfuura 2 μg.
A-1 Mhando yemuenzaniso yakanyanya kukura
- Deredza sampuro yemari.
A-2 Mimwe mienzaniso ine yakakwira DNA yezviri mukati uye inogona kurapwa neDNase.
- Ita RNase-Yemahara DNase kurapwa kune yakawanikwa RNA mhinduro, uye iyo RNA inogona kushandiswa zvakananga kuzviedza zvinotevera mushure mekurapwa, kana inogona kuvhenekerwa neRNA yekuchenesa makiti.
Kune magirazi egirazi, akabikwa pa150 ° C kwe4 h. Pamidziyo yepurasitiki, yakanyudzwa mu0.5 M NaOH kwemaminetsi gumi, ndokunyatsogezwa nemvura isina RNase uye wozadza kuti ubvise zvachose RNase. Iwo ma reagents kana mhinduro dzinoshandiswa mukuyedza, kunyanya mvura, dzinofanirwa kunge dzisina RNase. Shandisa RNase-yemahara mvura kune ese reagent gadziriro (wedzera mvura kune yakachena girazi bhodhoro, wedzera DEPC kune yekupedzisira fungidziro ye0.1% (V / V), zunza husiku uye autoclave).
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