FAQ's donKit ɗin keɓewar dabbobi na jimlar RNA

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Ba a fitar da RNA ko amfanin RNA yayi ƙasa

Sau da yawa akwai dalilai iri-iri waɗanda ke shafar ingancin farfadowa, kamar: samfurin nama na RNA abun ciki, hanyar aiki, ƙarar haɓaka, da sauransu.

1. Ice wanka ko cryogenic (4 ° C) centrifugation aka yi a lokacin aiki.

Shawarwari: Yi aiki a dakin da zafin jiki (15-25 ° C) a duk tsawon tsari, kada ku yi wanka da centrifuge a ƙananan yanayin zafi.

2. Ƙimar samfurin da ba daidai ba ko yawan lokacin ajiyar samfurin.

Shawarwari: Ajiye samfurori a -80 ° C ko daskare a cikin ruwa nitrogen kuma kauce wa daskare-narke maimaita amfani;yi ƙoƙarin amfani da sabobin nama ko ƙwayoyin al'ada don hakar RNA.

3. Rashin isasshen samfurin lysis.

Shawarwari: Lokacin da aka haɗa nama, tabbatar da cewa nama ya isa daidai kuma cewa ƙwayoyin nama sun rabu sosai don bayyana sakin RNA.

4. Ba a ƙara eluent daidai ba.

Shawarwari: Tabbatar da cewa RNase-Free ddH₂O ana ƙara juzu'i zuwa tsakiyar membrane ginshiƙin tsarkakewa.

5. Ba a ƙara madaidaicin ƙarar cikakken ethanol zuwa Buffer RL2 ko Buffer RW2 ba.

Shawarwari: Bi umarnin, ƙara madaidaicin ƙarar cikakkar ethanol zuwa Buffer RL2 da Buffer RW2 kuma gauraya da kyau kafin amfani da kit ɗin.

6. Nama samfurin sashi bai dace ba.

Shawarwari: Yi amfani da 10-20 MG na nama ko (1-5) × 10⁶Kwayoyin a cikin 500 μl buffer RL1, saboda yawan amfani da nama zai iya haifar da raguwar hakar RNA.

7. Rashin ingantaccen ƙarar ƙyalli ko rashin cikawa.

Shawarwari: Girman haɓakar ginshiƙin tsarkakewa shine 50-200 μl;idan tasirin elution bai gamsar ba, ana ba da shawarar tsawaita lokacin sanyawa dakin zafin jiki bayan ƙara preheated RNase-Free ddH₂O, misali na 5-10 min.

8.Shafin tsarkakewa yana da ragowar ethanol bayan wankewar buffer RW2.

Shawarwari: Idan akwai ragowar ethanol bayan wankewar Buffer RW2, centrifugation mara amfani na 1min, lokacin aikin centrifugation na bututu mara kyau za'a iya ƙara zuwa 2min, ko kuma ana iya sanya ginshiƙin tsarkakewa a dakin da zafin jiki na 5 min don cire isasshen ethanol.

RNA mai tsafta ya lalace

Ingancin RNA da aka tsarkake yana da alaƙa da abubuwa kamar adana samfurin, gurɓataccen RNase, da magudi, da sauransu.

1. Ba a adana samfuran nama a cikin lokaci.

Shawarwari: Idan ba a yi amfani da samfurori na nama ko sel a cikin lokaci ba bayan tattarawa, nan da nan a adana a -80 ° C ko nitrogen mai ruwa.Don cire RNA, yi amfani da sabon samfurin nama ko tantanin halitta a duk lokacin da zai yiwu.

2. Maimaita daskare-narke samfuran nama.

Shawarwari: Lokacin adana samfuran nama, yana da kyau a yanke su kanana don adanawa, kuma a cire ɗaya daga cikin guntun lokacin amfani da su don guje wa daskare-narke samfurin da kuma lalata RNA.

3. Ana gabatar da RNase ko a'a sanye da safar hannu, abin rufe fuska, da sauransu yayin aikin.

Shawarwari: Gwajin cirewar RNA an fi yin su a cikin ɗakunan sarrafa RNA daban kuma an share teburin kafin gwajin.

Sanya safar hannu da abin rufe fuska yayin gwajin don rage lalata RNA wanda ya haifar da gabatarwar RNase.

4. Reagents sun gurbata da RNase yayin amfani.

Shawarwari: Sauya da sabon Kit ɗin Keɓewar Dabbobi na RNA don gwaje-gwaje masu alaƙa.

5. Bututun centrifuge, tukwici, da sauransu da ake amfani da su wajen sarrafa RNA sun gurɓata da RNase.

Shawarwari: Tabbatar da cewa bututun centrifuge, tukwici, pipettes, da sauransu. da ake amfani da su wajen cire RNA duk ba su da RNase.

RNA da aka tsarkake yana shafar gwaje-gwaje na ƙasa

RNA da aka tsarkake ta hanyar ginshiƙin tsarkakewa, idan ions gishiri, abun cikin furotin ya yi girma zai shafi gwajin ƙasa, kamar: juzu'i na baya, Northern Blot et al.

1. RNA da aka elutioned yana da ragowar ion gishiri.

Shawarwari: Tabbatar da cewa an ƙara madaidaicin ƙarar ethanol zuwa Buffer RW2 kuma kuyi ginshiƙan tsarkakewa guda 2 a saurin centrifugal da aka nuna don aiki;idan akwai ragowar ion gishiri, bar ginshiƙin tsarkakewa zuwa Buffer RW2 na 5 min a zafin daki kuma yi centrifugation don haɓaka kawar da gurɓataccen gishiri.

2. Ethanol saura a cikin elutioned RNA.

Shawarwari: Tabbatar da cewa bayan buffer RW2 wankewa, yi aikin centrifugation mara amfani a cikin saurin centrifugation da aka nuna don aiki, ƙara lokacin aikin centrifugation mara amfani zuwa 2 min idan har yanzu akwai ragowar ethanol, ko barin shi a cikin dakin da zafin jiki na 5 min bayan fanko tube centrifugation don ƙara yawan cirewar ethanolue.

Warewa nau'ikan samfurin nulceic acid

Kayan keɓewar RNA na Foregene na iya samun jimillar keɓewa / cirewar RNA daga miya mai zuwa: Naman dabba, shuka, sel, hoto mai hoto, jini, da sauransu.

Yaya zan adana kayan

Ajiye zafin jiki na tsawon watanni 24.