I-RNase ligama elinovakalelo abangafuni ukuliva abafundi abaninzi abasoloko besenza imifuniselo yokutsalwa kwe-RNA.Ixhobe ngokupheleleyo, i-RNA eyathi ekugqibeleni yakhutshwa kunye ne-reagents enetyhefu kakhulu phenol kunye ne-chloroform yathotywa.Andixolelwanga!!!Namhlanje, makhe sijonge imvelaphi yeRnase edumileyo.

I-Ribonuclease (i-RNase), okanye i-RNase, yi-nuclease ekwazi ukukhupha i-RNA kwi-molecule encinci.I-RNase, njengeprotein encinci yemolekyuli, izinzile ngokungaqhelekanga .Ubushushu obuphezulu obuqhelekileyo kunye noxinzelelo oluphezulu lwe-sterilization yomphunga kunye ne-protein inhibitors ayikwazi ukuyivula ngokupheleleyo.Ukuzinza kwe-RNase ikakhulu kuvela kwiibhondi ze-disulfide kwisakhiwo.Umzekelo, i-RNase ye-pancreas esetyenziswa ngokuqhelekileyo ine-amino acids ezili-124 kuphela, kodwa iqulethe iibhondi ze-disulfide ezi-4.Ibhondi yesulfure kunye ne-disulfide bond inika i-RNase ngozinzo olubalaseleyo lobushushu.Ukongeza, i-rnase inobunzima obuncinci bemolekyuli kwaye inokubuyisela ngokukhawuleza ukuhambelana kwayo kwasekuqaleni kwiimeko ezininzi.

Ukongeza ekubeni uzinze kakhulu,Ii-RNases zikho yonke indawo kwilabhoratri .I-RNase yindlela yokukhusela yebhayoloji.Kwiseli, i-RNA yangaphandle idla ngokubulala.Xa ithelekiswa ne-DNA yangaphandle, i-RNA yangaphandle idla ngokuba yingozi ngakumbi.I-RNA ibhalwe ngovuyo kwaye iguqulelwe, ngoko ke phantse zonke izinto eziphilayo ziye zavela kwi-RNases ukukhusela kuhlaselo lwe-RNA yangaphandle.Ke ngoko, iiseli zebhaktheriya ezilinywa kwilabhoratri kwaye wena ukhupha i-RNA ukhupha ivumba le-RNase.Ulwelo lomzimba womntu (amathe, iinyembezi, njl.njl.) luqulethe isixa esikhulu se-RNase, ngoko musa ukukhala xa i-RNA ithotywa.Okukhona ukhala, kokukhona ukuwohloka kweRNA kubi!!UDade Daiyu akafanelanga ukutsalwa kwe-RNA!

Ukongeza, ulusu lwakho oluthambileyo lukwaqulethe i-RNase eninzi, kwaye iziphawuli, iipayipi, iingcango zesikhenkcisi, kunye nezibambo zocango ezichukunyiswe lusu nazo zine-RNase.

Ngokugxalathelana okungaka, makhe sijonge indlela yokujongana nee-RNases.

Into yokuqala ecingwa ngumntu wonke xa esusa iRNAI-DEPC(i-diethyl pyrocarbonate).I-DEPC ikakhulu i-denatures iprotheni ngokudibanisa ne-imidazole iringi yeqela elisebenzayo le-RNase i-histidine, ngaloo ndlela inqanda umsebenzi we-enzyme.I-0.1% ye-DEPC ingaba nefuthe elingcono lokususa kwi-Rnase, kodwa kufuneka sinikele ingqalelo ukuba i-DEPC i-carcinogen eyaziwayo, ngoko ke kufuneka kuhlawulwe ingqalelo ekhethekileyo xa usebenzisa.

Kwi-RNase, kufuneka siqale kwimiba emibini,eyokuqala kukuthintela umsebenzi we-endogenous RNase

Ii-reagents ze-RNA eziqhelekileyo ezifana ne-guanidine isothiocyanate kunye ne-DTT equlethwe kwi-Trizol inokuvula i-disulfide bond ye-RNase, kodwa kusekho ezinye ii-RNases, ngakumbi kwiisampuli zezicubu, ngoko ke nikela ingqalelo kubushushu obuphantsi.

1.Ngokukhawuleza untywilisele isampuli yezicubu kwi-nitrogen engamanzi emva kokuyikhupha, okanye kwisisombululo sokugcinwa kwe-RNA yorhwebo.

2.Emva kokukhupha i-RNA yesampuli yeseli, yongeza kwisisombululo se-lysis kwaye uyibeke kwibhokisi yeqhwa

3. Kungcono ukusebenzisa initrogen engamanzi xa ucola xa iisampulu zethishu zihomogenized.Xa usebenzisa i-homogenizer yombane ngaphandle kwe-nitrogen engamanzi, nikela ingqalelo ekupholiseni ngokupheleleyo i-adapter ye-homogenate.

Eyesibini yi-DNase yangaphandle

1.Zixhobe ngokupheleleyo, nxiba idyasi yelebhu, nxiba imaski, kwaye uqiniseke ukuba unxibe ipere yeeglavu ezintsha (musa ukonga kakhulu!! Kufuneka kuqatshelwe ukuba iTrizol idleka kakhulu, kwaye yomelele kakhulu ngeeglavu, ngoko ke musa ukuthontsiza ezandleni zakho).

2.Zonke iingcebiso ezisetyenzisiweyo ze-pipette, iityhubhu ze-EP, iityhubhu ze-PCR kunye nezinye izixhobo kufuneka ziphathwe nge-de-RNase.Inokuthi ifakwe kwi-0.1% DEPC kwaye emva koko icinezelwe phantsi koxinzelelo oluphezulu.Nika ingqalelo kuqhaqho kwi-fume hood.Oozwilakhe basekhaya banokuthenga ngokuthe ngqo izinto ezityiwayo zokususa ii-enzymes.

PS: Makhe ndikuxelele indlela eyonqenayo.Nangona ukushisa okuphezulu kunye noxinzelelo oluphezulu alukwazi ukususa ngokupheleleyo i-RNase, iya kususa inxalenye enkulu yayo.Amaxesha ama-2 obushushu obuphezulu kunye noxinzelelo oluphezulu lunefuthe elihle, kwaye ukukhutshwa kwe-RNA kunempembelelo encinci.

3.Utywala bunokukhupha iiproteni,ngoko itheyibhile yokukhutshwa kwe-RNA inokusulwa nge-75% yotywala , kunye neeglavu nazo zinokutshizwa ngotywala.

4.Isisombululo sokugqibela se-RNA kunye netyhubhu ye-centrifuge kufuneka kwakhona i-de-RNse iphathwe.Amanzi e-DEPC sisisombululo esiqhelekileyo sokunyibilikisa i-RNA.Makhe sithethe ngendlela echanekileyo yokulungiselela amanzi e-DEPC (khumbula ukuphinda upakishe i-DEPC yorhwebo xa uyithenga)

Yongeza i-DEPC kumanzi e-ultrapure kwi-1: 1000, xubha kakuhle, makube ubusuku bonke kwi-37 ° C, kwaye inzalo kwi-121 ° C phantsi kobushushu obuphezulu kunye noxinzelelo oluphezulu lwemizuzu eyi-15.Amanzi e-DEPC anokugcinwa kwi--20 ° C kwi-aliquots ye-1ml.

Ekugqibeleni, ukushwankathela: ubushushu obuphantsi ngundoqo, izinto ezisetyenziswayo ziphathwa kakuhle, zixhobe ngokupheleleyo kwaye zincinci ukuthetha!

Ewe, yiloo nto yeqhinga lanamhlanje.Ndikunqwenelela lonke ugxininiso lwe-RNA kunye nobunyulu obukhankanyiweyo.Zombini i-A260/A280 ziyi-2.0!!!

Ngokuqinisekileyo, ukuba usebenzisa iubushushu begumbi ukusebenza RNA ikhithi yokutsalwa, usenokungadibani nezi ngxaki zingasentla.

Ukusebenza kwiqondo lokushisa, ngaphandle kokongeza i-DNase, ikhupha i-RNA iyonke kwiiseli kwimizuzu eyi-11, kwaye ikhuphe i-RNA iyonke kwizicubu zezilwanyana okanye izityalo kwimizuzu engama-30.

Ngeisampulu zolingo, nceda uqhagamshelane:overseas@foregene.com

Iimveliso ezinxulumeneyo:

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/