RNase ndi mawu ovuta omwe ophunzira ambiri omwe nthawi zambiri amachita zoyeserera za RNA safuna kumva.Pokhala ndi zida zonse, RNA yomwe pamapeto pake idatulutsidwa ndi ma reagents oopsa kwambiri a phenol ndi chloroform idawonongeka.Sindinayanjanenso!!!Lero, tiyeni tione chiyambi cha Rnase wotchuka.

Ribonuclease (RNase), kapena RNase, ndi nuclease yomwe imatha hydrolyze RNA kukhala mamolekyu ang'onoang'ono.RNase, monga puloteni yaing'ono ya molekyulu, imakhala yokhazikika modabwitsa .Kutentha kokhazikika komanso kuthamanga kwambiri kwa nthunzi yotseketsa komanso zoletsa mapuloteni sangathe kuzimitsa.Kukhazikika kwa RNase makamaka kumachokera ku zomangira za disulfide mu kapangidwe kake.Mwachitsanzo, RNase yomwe imagwiritsidwa ntchito kwambiri ya kapamba ya bovine ili ndi ma amino acid 124 okha, koma imakhala ndi zomangira 4 za disulfide.Sulfur bond ndi disulfide bond zimapatsa RNase kukhazikika kwamafuta.Kuonjezera apo, rnase ili ndi kulemera kochepa kwa maselo ndipo imatha kubwezeretsanso mawonekedwe ake oyambirira nthawi zambiri.

Kuphatikiza pa kukhala wokhazikika kwambiri,Ma RNases amapezeka paliponse mu labotale .RNase ndi njira yoteteza zachilengedwe.Kwa selo, RNA yakunja nthawi zambiri imakhala yakupha.Poyerekeza ndi DNA yakunja, RNA yakunja nthawi zambiri imakhala yowopsa.RNA imalembedwa mosangalala ndikumasuliridwa, kotero pafupifupi zamoyo zonse zasintha ma RNase kuti ziteteze ku kuukira kwa RNA yachilendo.Chifukwa chake, ma cell a bakiteriya omwe amalimidwa mu labotale ndipo inu omwe mumatulutsa RNA mumatulutsa fungo la RNase.Madzi a m’thupi la munthu (malovu, misozi, ndi zina zotero) ali ndi RNase yambiri, choncho musalire pamene RNA ikuwonongeka.Mukalira kwambiri, m'pamenenso kuwonongeka kwa RNA kumayipitsitsa!!Mlongo Daiyu sali woyenera kutulutsa RNA!

Kuphatikiza apo, khungu lanu losakhwima limakhalanso ndi RNase yambiri, ndipo zolembera, ma pipette, zitseko za firiji, ndi zogwirira zitseko zomwe zakhudzidwa ndi khungu zimakhalanso ndi RNase.

Ndi kuyendayenda kochuluka, tiyeni tiwone momwe tingachitire ndi RNases.

Chinthu choyamba chimene aliyense amaganiza pochotsa RNA ndiDEPC(diethyl pyrocarbonate).DEPC makamaka denatures mapuloteni kaphatikizidwe ndi imidazole mphete ya RNase yogwira gulu histidine, potero inhibiting ntchito ya puloteni.0.1% DEPC ikhoza kukhala ndi zotsatira zabwino zochotsa pa Rnase, koma tiyenera kumvetsera kuti DEPC ndi carcinogen yodziwika bwino, kotero chisamaliro chapadera chiyenera kuperekedwa pochigwiritsa ntchito.

Kwa RNase, tiyenera kuyambira mbali ziwiri,choyamba ndi kuletsa ntchito amkati RNase

Ochiritsira RNA m'zigawo reagents monga guanidine isothiocyanate ndi DTT ali Trizol akhoza kutsegula disulfide chomangira cha RNase, koma pali RNases ena, makamaka zitsanzo minofu, kotero kulabadira kutentha otsika.

1.Nthawi yomweyo miza minofuyo mu nayitrogeni wamadzimadzi mutatha kuitulutsa, kapena mu njira yosungiramo malonda a RNA.

2.Pambuyo pochotsa RNA yachitsanzo cha selo, yonjezerani ku njira ya lysis ndikuyiyika pa ayezi

3. Ndi bwino kugwiritsa ntchito nayitrogeni wamadzimadzi pogaya pamene minofu zitsanzo ndi homogenized.Mukamagwiritsa ntchito homogenizer yamagetsi popanda nayitrogeni wamadzimadzi, tcherani khutu kuti muzitha kuziziritsa adapter ya homogenate.

Yachiwiri ndi exogenous DNase

1.Khalani ndi zida zonse, valani malaya a labu, valani chigoba, ndipo onetsetsani kuti mwavala magolovesi atsopano (musakhale osunga zinthu kwambiri!! Dziwani kuti Trizol ndi yowononga kwambiri, komanso imakhala yamphamvu kwambiri kudzera m'magolovesi, kotero musadonthe m'manja mwanu).

2.Malangizo onse ogwiritsidwa ntchito a pipette, machubu a EP, machubu a PCR ndi zida zina ziyenera kuthandizidwa ndi de-RNase.Itha kuviikidwa mu 0.1% DEPC kenako kukakamizidwa ndi kupsinjika kwakukulu.Samalani ntchito mu fume hood.Olamulira ankhanza am'deralo amatha kugula mwachindunji zinthu zochotsera ma enzyme.

PS: Ndikuuzeni njira yaulesi.Ngakhale kutentha kwambiri komanso kuthamanga kwambiri sikungathe kuchotsa RNase, kumachotsa gawo lalikulu.Nthawi za 2 za kutentha kwakukulu ndi kuthamanga kwakukulu kumakhala ndi zotsatira zabwino, ndipo kuchotsa kwa RNA kumakhala ndi zotsatira zochepa.

3.Mowa ukhoza kusokoneza mapuloteni,kotero tebulo lochotsa RNA likhoza kupukuta ndi mowa wa 75%. , ndi magolovesi amathanso kupopera mowa.

4.Njira yomaliza ya RNA yosungunuka ndi chubu la centrifuge iyeneranso kuthandizidwa ndi de-RNase.Madzi a DEPC ndi njira yomwe imagwiritsidwa ntchito kwambiri ndi RNA.Tiyeni tikambirane njira yolondola yokonzekera madzi a DEPC (kumbukirani kukonzanso DEPC yamalonda mukagula)

Onjezani DEPC kumadzi a ultrapure pa 1: 1000, gwedezani bwino, lolani kuyimirira usiku wonse pa 37 ° C, ndikutenthetsa pa 121 ° C pansi pa kutentha kwakukulu ndi kuthamanga kwakukulu kwa mphindi 15.Madzi a DEPC amatha kusungidwa pa -20 ° C mu 1ml aliquots.

Pomaliza, kunena mwachidule: kutentha kochepa ndiye fungulo, zogwiritsidwa ntchito zimasamalidwa bwino, zida zonse komanso zolankhula zochepa!

Chabwino, ndizo za njira yamasiku ano.Ndikufunirani inu nonse ndende ya RNA ndi chiyero chomwe mwatchula.Onse A260/A280 ndi 2.0!!!

Inde, ngati mugwiritsa ntchito aChipinda chogwiritsira ntchito kutentha kwa RNA, simungakumane ndi mavuto omwe ali pamwambawa.

Opaleshoni kutentha kwa firiji, popanda kuwonjezera DNase, imatulutsa RNA yonse m'maselo mumphindi 11, ndikuchotsa RNA yonse kuchokera kuzinthu zanyama kapena zomera mumphindi 30.

Kwa zitsanzo zoyeserera, lemberani:overseas@foregene.com

Zogwirizana nazo:

https://www.foreivd.com/cell-total-rna-isolation-kit-product/

https://www.foreivd.com/animal-total-rna-isolation-kit-product/

https://www.foreivd.com/plant-total-rna/