FAQ zaAnimal Total RNA Isolation zida

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RNA sinachotsedwe kapena zokolola za RNA ndizochepa

Nthawi zambiri pamakhala zinthu zosiyanasiyana zomwe zimakhudza kuchira bwino, monga: minofu ya RNA yokhutira, njira yogwirira ntchito, voliyumu ya elution, ndi zina zambiri.

1. Kusambira kwa ayezi kapena cryogenic (4 ° C) centrifugation kunachitika panthawi ya ntchito.

Malangizo: Gwiritsani ntchito kutentha (15-25 ° C) panthawi yonseyi, osasamba madzi oundana ndi centrifuge pa kutentha kochepa.

2. Kusungidwa kwachitsanzo kosayenera kapena nthawi yochuluka yosungira zitsanzo.

Malangizo: Sungani zitsanzo pa -80 ° C kapena kuzizira mu nayitrogeni wamadzimadzi ndipo pewani kugwiritsa ntchito kuzizira kobwerezabwereza;yesetsani kugwiritsa ntchito minofu yatsopano kapena maselo otukuka kuti muchotse RNA.

3. Kusakwanira kwachitsanzo lysis.

Malangizo: Mukamagwirizanitsa minofu, onetsetsani kuti minofuyo ili ndi homogenized mokwanira komanso kuti maselo a minofu agawanika mokwanira kuti afotokoze kutulutsidwa kwa RNA.

4. Chosowacho sichiwonjezedwa bwino.

Malangizo: Tsimikizirani kuti RNase-Free ddH₂O amawonjezedwa pang'onopang'ono pakati pa nembanemba yoyeretsa.

5. Voliyumu yolondola ya ethanol yokwanira sinawonjezedwe ku Buffer RL2 kapena Buffer RW2.

Malangizo: Tsatirani malangizo, onjezani voliyumu yolondola ya ethanol ku Buffer RL2 ndi Buffer RW2 ndikusakaniza bwino musanagwiritse ntchito zida.

6. Mlingo wa zitsanzo za minofu siwoyenera.

Malangizo: Gwiritsani ntchito 10-20 mg ya minofu kapena (1-5) × 10⁶ma cell pa 500 μl buffer RL1, chifukwa kugwiritsa ntchito minofu mopitirira muyeso kungayambitse kuchepa kwa RNA.

7. Voliyumu yolakwika kapena kusakwanira.

Malangizo: Voliyumu ya elution ya gawo loyeretsa ndi 50-200 μl;ngati mphamvu ya elution siili yokhutiritsa, tikulimbikitsidwa kuwonjezera nthawi yoyika kutentha kwa chipinda mutawonjezera RNase-Free ddH yotenthetsera.₂O, mwachitsanzo kwa 5-10 min.

8.Mzere woyeretsedwa uli ndi zotsalira za ethanol pambuyo pa kusamba kwa Buffer RW2.

Malangizo: Ngati pali zotsalira za ethanol pambuyo pa kusamba kwa Buffer RW2, chubu chopanda kanthu centrifugation kwa 1min, nthawi yopanda kanthu chubu centrifugation ntchito akhoza ziwonjezeke kwa 2min, kapena ndime kuyeretsa akhoza kuikidwa pa firiji kwa mphindi 5 mokwanira kuchotsa Mowa yotsalira.

RNA yoyeretsedwa ndiyonyozeka

Ubwino wa RNA yoyeretsedwa ikugwirizana ndi zinthu monga kusungidwa kwa chitsanzo, kuipitsidwa kwa RNase, ndi kusintha, ndi zina zotero.

1. Zitsanzo za minofu sizimasungidwa nthawi.

Malangizo: Ngati zitsanzo za minofu kapena maselo sagwiritsidwa ntchito munthawi yake mutatolera, nthawi yomweyo sungani pa -80 °C kapena nayitrogeni wamadzimadzi.Kuti mutulutse RNA, gwiritsani ntchito minofu yongotengedwa kumene kapena maselo a cell ngati kuli kotheka.

2. Kuzizira kobwerezabwereza kwa zitsanzo za minofu.

Malangizo: Posunga zitsanzo za minofu, ndi bwino kuzidula kuti zisungidwe, ndikuchotsa chimodzi mwa zidutswazo pozigwiritsira ntchito kuti musamaundane mobwerezabwereza ndi kuwonongeka kwa RNA.

3. RNase imayambitsidwa kapena kusavala magolovesi otayika, masks, ndi zina zotero panthawi ya opaleshoni.

Malangizo: Kuyesa kwa RNA m'zigawo kumachitika bwino m'zipinda zosinthira za RNA ndipo tebulo limachotsedwa kuyesako kusanachitike.

Valani magolovesi ndi masks otayika panthawi yoyesera kuti muchepetse kuwonongeka kwa RNA komwe kumachitika chifukwa cha kukhazikitsidwa kwa RNase.

4. Ma reagents amadetsedwa ndi RNase pakugwiritsa ntchito.

Malangizo: M'malo ndi Animal Total RNA Isolation Kit yatsopano yoyeserera yofananira.

5. Machubu a centrifuge, nsonga, ndi zina zotere zomwe zimagwiritsidwa ntchito pakusintha kwa RNA zimaipitsidwa ndi RNase.

Malangizo: Tsimikizirani kuti machubu a centrifuge, nsonga, mapaipi, ndi zina zomwe zimagwiritsidwa ntchito pochotsa RNA zonse ndi Zaulere za RNase.

Oyeretsedwa analandira RNA amakhudza zoyesera kumunsi

RNA yoyeretsedwa ndi ndime yoyeretsera, ngati ayoni amchere, zomanga thupi ndi zazikulu kwambiri zidzakhudza kuyesera kumtunda, monga: reverse transcription,Northern Blot et al.

1. The elutioned RNA ili ndi zotsalira za ayoni zamchere.

Malangizo: Tsimikizirani kuti voliyumu yolondola ya ethanol yawonjezedwa ku Buffer RW2 ndikuchita ndime zoyeretsera 2 pa liwiro la centrifugal lomwe lasonyezedwa kuti ligwire ntchito;ngati pali zotsalira za mchere wa ion, siyani mzere woyeretsera ku Buffer RW2 kwa mphindi 5 pa kutentha kwapakati ndikuchita centrifugation kuti muwonjezere kuchotsa kuipitsidwa kwa mchere.

2. Ethanol zotsalira mu elutioned RNA.

Malangizo: Tsimikizirani kuti mutatha kutsuka kwa bafa RW2, gwiritsani ntchito chubu cha centrifugation chopanda kanthu pa liwiro la centrifugation lomwe likuwonetsedwa kuti ligwire ntchito, onjezerani nthawi ya ntchito yopanda kanthu ya chubu centrifugation mpaka 2 min ngati pali zotsalira za ethanol, kapena musiye kutentha kwa mphindi 5 pambuyo pa chubu chopanda kanthu centrifugation kuti muwonjezere kuchotsedwa kwa ethanol.

Kupatula mitundu ya zitsanzo za nulceic acid

Zida za Foregene's RNA Isolation zimatha kupeza kudzipatula/kuchotsa kwathunthu kwa RNA kuchokera ku zitsanzo zotsatirazi: minofu ya nyama, chomera, maselo, ma virus, magazi, ndi zina.

Ndisunga bwanji zida

Kusungirako kutentha kwa chipinda kwa miyezi 24.