ⅠNucleic acidiisingeniso
I-Nucleic acid ihlukaniswe yaba yi-deoxyribonucleic acid (DNA) kanye ne-ribonucleic acid (RNA), phakathi kwayo i-RNA ihlukaniswe yaba yi-ribosomal RNA(rRNA), isithunywa i-RNA(mRNA) futhi idlulisela i-RNA(tRNA) ngokwemisebenzi yayo ehlukene.I-DNA igxile kakhulu ku-nucleus, mitochondria ne-chloroform, kuyilapho i-RNA isatshalaliswa kakhulu ku-cytoplasm.Njengesisekelo esibonakalayo sokuvezwa kofuzo, isizinda se-nucleic acid sidlala indima ebaluleke kakhulu ocwaningweni lwebhayoloji yamangqamuzana kanye nokuxilongwa kwamangqamuzana omtholampilo.Ukugxila nokuhlanzeka kwesizinda se-nucleic acid kuzothinta ngokuqondile i-PCR elandelayo, ukulandelana, ukwakhiwa kwe-vector, ukugaya kwe-enzyme nokunye ukuhlola.
Ⅱ Isizinda se-Nucleic acid kanye nendlela yokuhlanza
① Indlela yokukhipha i-Phenol/chloroform
I-Phenol/chloroform extraction iyindlela yakudala yokukhipha i-DNA, esebenzisa ngokuyinhloko izinyibilikisi ezimbili ezihlukene zemvelo ukwelapha amasampula, ukuncibilikisa i-DNA esekelwe ku-nucleic acid esigabeni samanzi, ama-lipids esigabeni sezinto eziphilayo, kanye namaprotheni phakathi kwezigaba ezimbili.Le ndlela inezinzuzo zezindleko eziphansi, ukuhlanzeka okuphezulu kanye nomphumela omuhle.Ukungalungi ukusebenza okuyinkimbinkimbi futhi isikhathi eside.
② Indlela ye-Trizol
Indlela ye-Trizol iyindlela yakudala yokukhipha i-RNA.Indlela ye-Trizol ihlukaniswe yaba yisigaba se-aqueous kanye nesigaba se-organic ngemva kwe-centrifugation ne-chloroform, lapho i-RNA ichithwa esigabeni samanzi, isigaba se-aqueous sidluliselwa ku-tube entsha ye-EP, imvula itholakala ngemva kokwengeza isopropanol, bese i-ethanol purification.Le ndlela ifaneleka ekukhishweni kwe-RNA ezicutshini zezilwane, amaseli namagciwane.
③ Indlela yokuhlanza ikholomu ye-Centrifugal
Indlela yokuhlanza ikholomu ye-Centrifuge ingakhangisa i-DNA ngokukhethekile ngokusebenzisa izinto ezikhethekile ze-silicon matrix adsorption, kuyilapho i-RNA nephrotheni ingadlula kahle, bese isebenzisa usawoti omningi ophansi we-PH ukuhlanganisa i-nucleic acid, usawoti ophansi ophezulu we-PH value elution ukuze uhlukanise futhi uhlanze i-nucleic acid.Izinzuzo ukuhlushwa okuphezulu kokuhlanza, ukuzinza okuphezulu, asikho isidingo se-solvent ye-organic, nezindleko eziphansi.Ububi ukuthi idinga ukufakwa i-centrifuged isinyathelo ngesinyathelo, izinyathelo eziningi zokusebenza.
④ Indlela yobuhlalu kazibuthe
Indlela yobuhlalu kazibuthe iwukuhlukanisa isampula yethishu yeseli nge-lysate, kudedelwe i-nucleic acid kusampula, bese ama-molecule e-nucleic acid akhangiswe ngokuqondile ebusweni bobuhlalu obuzibuthe, kuyilapho ukungcola okunjengamaphrotheni noshukela kushiywa phakathi. uketshezi.Ngezinyathelo zokuhlukaniswa kwezicubu zamangqamuzana, ukubopha ubuhlalu obuzibuthe nge-nucleic acid, ukugeza kwe-nucleic acid, i-nucleic acid elution, njll., ekugcineni kutholakala i-nucleic acid emsulwa.Izinzuzo ziwukusebenza okulula nokusebenzisa isikhathi esifushane, ngaphandle kwesidingo sesinyathelo se-centrifugation.Inezidingo eziphansi zobuchwepheshe futhi ingakwazi ukubona ukusebenza okuzenzakalelayo nobuningi.Inhlanganisela ethize yobuhlalu obuzibuthe kanye ne-nucleic acid yenza i-nucleic acid ekhishiwe ibe nokugxila okuphezulu nobumsulwa.Ububi ukuthi intengo yemakethe yamanje ibiza kakhulu.
⑤ Ezinye izindlela
Ngaphezu kwalezi zindlela ezine ezingenhla, kukhona ukuqhekeka abilayo, indlela kasawoti egxilile, indlela yokuhlanza i-anionic, indlela ye-ultrasonic kanye nendlela ye-enzymatic, njll.
Ⅲ Uhlobo lokukhishwa kwe-nucleic acid
I-Foregene inenkundla ehamba phambili emhlabeni ye-Direct PCR, inkundla ye-RNA Isolation enamakholomu amabili (DNA-kuphela + RNA kuphela ).Imikhiqizo eyinhloko ihlanganisa i-DNA/RNA isolation kits, i-PCR kanye ne-Direct PCR reagents yama-molecular lab reagents series.
① Isamba sokukhishwa kwe-RNA
Isamba samasampula okukhishwa kwe-RNA ahlanganisa igazi, amangqamuzana, izicubu zezilwane, izitshalo, amagciwane, njll. Ukuhlanzeka okuphezulu kanye nokugxiliswa okuphezulu kwengqikithi ye-RNA kungatholwa ngokukhishwa okuphelele kwe-RNA, engasetshenziswa ku-RT-PCR, ukuhlaziywa kwe-chip, ukuhumusha kwe-in vitro, i-molecular cloning, i-Dot Blot nezinye izivivinyo.
Okuhlobene ne-ForegeneI-RNA Isolation Kits
Ikhithi Yokuhlukanisa Yezilwane Ingqikithi ye-RNA--Khipha ngokushesha nangempumelelo ukuhlanzeka okuphezulu nekhwalithi ephezulu ingqikithi ye-RNA ezicutshini zezilwane ezahlukahlukene.
Ikhithi Yokuzihlukanisa Yeseli Ye-RNA--I-RNA ehlanzwe kakhulu futhi yekhwalithi ephezulu ingatholakala kumaseli ahlukahlukene akhuliswe ngemizuzu eyi-11.
Ikhithi Yokuhlukanisa Yezitshalo Isamba se-RNA--Khipha ngokushesha i-RNA yekhwalithi ephezulu kumasampula ezitshalo ane-polysaccharide ephansi nokuqukethwe kwe-polyphenol.
Ikhithi yokuhlukanisa i-Viral RNA--Hlukanisa futhi uhlanze ngokushesha i-RNA yegciwane kumasampula afana ne-plasma, i-serum, uketshezi lomzimba olunganamaseli kanye nezinto ezingaphezu kwamandla amaseli.
② Ukukhishwa kwe-Genomic DNA
Amasampula okukhishwa kwe-Genomic DNA ahlanganisa inhlabathi, indle, igazi, amaseli, izicubu zezilwane, izitshalo, amagciwane, njll. Isizinda se-Genomic DNA singasetshenziswa ekugayweni kwe-enzyme, ukwakhiwa komtapo wezincwadi we-DNA, i-PCR, ukulungiswa kwe-antibody, ukuhlaziywa kwe-Western blot hybridization, i-gene chip, ephezulu. -ukulandelana komphumela nokunye ukuhlola.
Okuhlobene ne-ForegeneI-DNA Isolation Kits
Ikhithi yokuhlukanisa i-DNA yezicubu zezilwane--Ukukhipha ngokushesha kanye nokuhlanzwa kwe-genomic DNA emithonjeni eminingi, njengezicubu zezilwane, amaseli, njll.
Ikhithi ye-DNA Midi yegazi (1-5ml)--Hlanza ngokushesha i-genomic DNA yekhwalithi ephezulu egazini eline-anticoagulated (1-5ml).
I-Buccal Swab/FTA Card DNA Isolation Kit--Hlanza ngokushesha i-DNA ye-genomic yekhwalithi ephezulu kumasampula e-buccal swab/FTA Card.
Ikhithi yokuhlukanisa i-Plant DNA--Hlanza ngokushesha futhi uthole i-DNA ye-genomic yekhwalithi ephezulu kumasampula ezitshalo (okuhlanganisa ama-polysaccharides namasampula ezitshalo ze-polyphenol)
③ Ukukhishwa kwe-Plasmid
I-Plasmid iwuhlobo lwe-molecule encane eyindilinga ye-DNA emangqamuzaneni, okuwumthwalo ovamile wokuhlanganiswa kabusha kwe-DNA.Indlela yokukhipha i-plasmid ukukhipha i-RNA, ukuhlukanisa i-plasmid ku-DNA yebhaktheriya ye-genomic, futhi kukhishwe amaprotheni nokunye ukungcola ukuze kutholwe i-plasmid emsulwa.
I-General Plasmid Mini Kit--Hlanza ngokushesha i-plasmid DNA yekhwalithi ephezulu kubhaktheriya eguquliwe ukuze uthole ukuhlolwa kwebhayoloji yamangqamuzana njengokuguqulwa nokugaya kwe-enzyme
④ Ezinye izinhlobo zokukhipha, isizinda se-miRNA, njll.
I-Animal miRNA Isolation Kit-- Khipha ngokushesha nangempumelelo izingcezu ezincane ze-RNA ze-20-200nt miRNA, i-siRNA, i-snRNA ezicutshini zezilwane ezihlukahlukene namaseli.
Ⅳ Izidingo zokukhishwa kwe-nucleic acid kanye nomphumela wokuhlanzas
① Ukuqinisekisa ubuqotho besakhiwo esiyinhloko se-nucleic acid.
② Yehlisa ukuphazamiseka kwamaprotheni, ushukela, ama-lipids namanye ama-macromolecules
③ Akufanele kube khona i-solvent ephilayo noma ukugcwala okuphezulu kwama-ion ensimbi okungavimbela i-enzyme kumasampuli e-nucleic acid.
④ I-RNA nokunye ukungcola kwe-nucleic acid kufanele kuqedwe lapho kukhishwa i-DNA, futhi ngokuphambene nalokho.
Isikhathi sokuthumela: Nov-24-2022
