I-RNase igama elibucayi abafundi abaningi abavame ukwenza izivivinyo zokukhipha i-RNA abafuni ukulizwa.Ihlome ngokugcwele, i-RNA eyagcina ikhishwe ngeziphehli ne-chloroform enobuthi kakhulu yehliswa.Angibuyiseli!!!Namuhla, ake sibheke umsuka we-Rnase edumile.

I-Ribonuclease (RNase), noma i-RNase, iyinuclease ekwazi ukwenza i-RNA i-hydrolyze ibe ama-molecule amancane.I-RNase, njengeprotheni ye-molecule encane, izinzile ngendlela engavamile .Izinga lokushisa elivamile elivamile kanye ne-high pressure sterilization yesitimu kanye nama-protein inhibitors awakwazi ukuwenza angasebenzi ngokuphelele.Ukuzinza kwe-RNase ikakhulukazi kuvela kumabhondi e-disulfide esakhiweni.Isibonelo, i-RNase evame ukusetshenziswa ye-bovine pancreas inama-amino acid ayi-124 kuphela, kodwa iqukethe amabhondi e-disulfide angu-4.Ibhondi yesibabule kanye nebhondi ye-disulfide inika i-RNase ngokuzinza okuhle kakhulu kwe-thermal.Ngaphezu kwalokho, i-rnase inesisindo esincane samangqamuzana futhi ingabuyisela ngokushesha ukufana kwayo kwasekuqaleni ezimweni eziningi.

Ngaphezu kokuzinza ngokwedlulele,Ama-RNase atholakala yonke indawo elabhorethri .I-RNase iyindlela yokuzivikela yebhayoloji.Engqamuzaneni, i-RNA yangaphandle ngokuvamile iyabulala.Uma kuqhathaniswa ne-DNA yangaphandle, i-RNA yangaphandle ivame ukuba yingozi kakhulu.I-RNA ilotshwe ngenjabulo futhi yahunyushwa, ngakho-ke cishe zonke izinto eziphilayo ziye zashintsha ama-RNas ukuze zivikele ekuhlaselweni kwe-RNA yangaphandle.Ngakho-ke, amangqamuzana ebhaktheriya atshalwa elabhorethri futhi wena okhipha i-RNA ukhipha iphunga le-RNase.Uketshezi lomzimba womuntu (amathe, izinyembezi, njll.) luqukethe inani elikhulu le-RNase, ngakho ungakhali lapho i-RNA yonakala.Lapho ukhala kakhulu, kubi kakhulu ukuwohloka kwe-RNA!!U-Dade Daiyu akafanelekele ukukhishwa kwe-RNA!

Ngaphezu kwalokho, isikhumba sakho esithambile futhi sine-RNase eningi, futhi izimpawu, amapayipi, iminyango yesiqandisi, nezibambo zeminyango ezithintwe isikhumba nazo zine-RNase.

Ngokugijima okungaka, ake sibheke ukuthi singabhekana kanjani nama-RNases.

Into yokuqala wonke umuntu ayicabangayo lapho esusa i-RNAI-DEPC(i-diethyl pyrocarbonate).I-DEPC ikakhulukazi iveza amaprotheni ngokuhlanganisa nendandatho ye-imidazole yeqembu elisebenzayo le-RNase i-histidine, ngaleyo ndlela ivimbele ukusebenza kwe-enzyme.I-0.1% DEPC ingaba nomthelela ongcono wokususa ku-Rnase, kodwa sidinga ukunaka ukuthi i-DEPC iyi-carcinogen eyaziwayo, ngakho-ke kufanele kuqashelwe ngokukhethekile uma uyisebenzisa.

Ku-RNase, sidinga ukuqala ezicini ezimbili,eyokuqala iwukuvimbela umsebenzi we-RNase engapheli

Ama-reagents ajwayelekile akhipha i-RNA njenge-guanidine isothiocyanate ne-DTT equkethwe ku-Trizol angavula ibhondi ye-disulfide ye-RNase, kodwa asekhona amanye ama-RNase, ikakhulukazi kumasampula ezicubu, ngakho-ke qaphela izinga lokushisa eliphansi.

1.Gxilisa ngokushesha isampula yethishu ku-nitrogen ewuketshezi ngemva kokuyikhipha, noma kusixazululo sokulondoloza i-RNA.

2.Ngemuva kokukhipha i-RNA yesampula yeseli, yengeze kusisombululo se-lysis bese uyiphonsa ebhokisini leqhwa

3. Kungcono ukusebenzisa i-nitrogen ewuketshezi ukugaya lapho amasampula ezicubu enziwa i-homogenized.Uma usebenzisa i-homogenizer kagesi ngaphandle kwe-nitrogen ewuketshezi, naka ukupholisa ngokugcwele i-adaptha ye-homogenate.

Eyesibili i-DNase yangaphandle

1.Zihlome ziphelele, gqoka ijazi laselabhu, gqoka imaski, futhi uqiniseke ukuthi ugqoka amagilavu ​​amasha (ungabongi kangako!! Kufanele kuqashelwe ukuthi i-Trizol igqwala kakhulu, futhi inamandla kakhulu ngokusebenzisa amagilavu, ngakho-ke ungaconsi ezandleni zakho).

2.Wonke amathiphu e-pipette asetshenzisiwe, amashubhu e-EP, amashubhu e-PCR nezinye izinto zokusebenza kufanele zilashwe de-RNase.Ingacwiliswa ku-0.1% DEPC bese icindezelwa ngaphansi kwengcindezi ephezulu.Naka ukuhlinza endaweni yokushisa.Omashiqela basendaweni bangathenga ngokuqondile izinto ezisetshenziswayo zokususa ama-enzyme.

PS: Ake ngikutshele indlela yobuvila.Nakuba izinga lokushisa eliphezulu nokucindezela okuphezulu akukwazi ukususa ngokuphelele i-RNase, izosusa ingxenye enkulu yayo.Izikhathi ezi-2 zokushisa okuphezulu nokucindezela okuphezulu kunomphumela omuhle, futhi ukukhishwa kwe-RNA kunomphumela omncane.

3.Utshwala bungadala amaprotheni,ngakho ithebula lokukhipha i-RNA lingasulwa ngotshwala obungama-75%. , namagilavu ​​nawo angafuthwa ngotshwala.

4.Isixazululo sokugcina sokuncibilika kwe-RNA kanye neshubhu ye-centrifuge kufanele futhi kuphathwe i-de-RNase.Amanzi e-DEPC iyisixazululo esivame ukusetshenziswa se-RNA.Ake sikhulume ngendlela efanele yokulungiselela amanzi e-DEPC (khumbula ukupakisha kabusha i-DEPC yokuhweba lapho uyithenga)

Faka i-DEPC emanzini e-ultrapure ngo-1: 1000, nyakazisa kahle, uyeke ume ubusuku bonke ku-37 ° C, bese ubulala inzalo ku-121 ° C ngaphansi kwezinga lokushisa eliphezulu kanye nokucindezela okuphezulu imizuzu engu-15.Amanzi e-DEPC angagcinwa ku -20°C ku-1ml aliquots.

Okokugcina, ukufingqa: izinga lokushisa eliphansi liyisihluthulelo, izinto ezidlekayo ziphathwa kahle, zihlome ngokuphelele futhi zincane ukukhuluma!

Hhayi-ke, yilokho okwesu lanamuhla.Ngikufisela konke ukugxila kwe-RNA nobumsulwa obashilo.Kokubili i-A260/A280 ingu-2.0!!!

Yiqiniso, uma usebenzisa aIkhithi yokukhipha i-RNA yegumbi lokushisa lokushisa, ungase ungahlangani nezinkinga ezingenhla.

Ukusebenza ekamelweni lokushisa, ngaphandle kokwengeza i-DNase, kukhipha i-RNA isiyonke kumaseli ngemizuzu engu-11, futhi kukhishwe isamba se-RNA ezicutshini zezilwane noma ezitshalweni ngemizuzu engama-30.

Ukuze uthole amasampula esilingo, sicela uthinte:overseas@foregene.com

Imikhiqizo Ehlobene:

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/