ⅠNucleic acidiintshayelelo
I-Nucleic acid ihlulwe ibe yi-deoxyribonucleic acid (DNA) kunye ne-ribonucleic acid (RNA), phakathi kwayo i-RNA yahlulwe ibe yi-ribosomal RNA (rRNA), isithunywa i-RNA (mRNA) kunye nokudlulisa i-RNA (tRNA) ngokwemisebenzi yayo eyahlukeneyo.I-DNA igxile kakhulu kwi-nucleus, mitochondria kunye ne-chloroform, ngelixa i-RNA isasazwa kakhulu kwi-cytoplasm.Njengesiseko sezinto ze-gene expression, i-nucleic acid extraction idlala indima ebaluleke kakhulu kuphando lwebhayoloji yemolekyuli kunye nokuxilongwa kweemolekyuli zeklinikhi.Ukugxininiswa kunye nokucoceka kwe-nucleic acid extraction kuya kuchaphazela ngokuthe ngqo i-PCR elandelayo, ukulandelelana, ukwakhiwa kwe-vector, ukugaya i-enzyme kunye nezinye iimvavanyo.
Ⅱ I-Nucleic acid extraction kunye nendlela yokucoca
① Indlela yokutsalwa kwePhenol/chloroform
I-Phenol / i-chloroform extraction yindlela ye-classical ye-DNA extraction, eyona nto isebenzisa i-solvents ezimbini ezahlukeneyo ze-organic ukunyanga iisampuli, ukuchithwa kwe-DNA esekelwe kwi-nucleic acid kwisigaba samanzi, i-lipids kwisigaba se-organic, kunye neeprotheni phakathi kwezigaba ezimbini.Le ndlela ineenzuzo zeendleko eziphantsi, ukucoceka okuphezulu kunye nomphumo omhle.Ukungalungi ngumsebenzi onzima kunye nexesha elide.
② Indlela yeTrizol
Indlela ye-Trizol yindlela yakudala yokutsalwa kwe-RNA.Indlela ye-Trizol ihlulwe kwisigaba se-aqueous kunye nesigaba se-organic emva kwe-centrifugation kunye ne-chloroform, apho i-RNA ichithwa kwisigaba samanzi, isigaba se-aqueous sidluliselwa kwi-tube entsha ye-EP, imvula ifumaneka emva kokongeza i-isopropanol, kwaye emva koko ukuhlanjululwa kwe-ethanol.Le ndlela ifanelekile ukukhutshwa kwe-RNA kwizicubu zezilwanyana, iiseli kunye neebhaktheriya.
③ Indlela yokucoca ikholamu ye-Centrifugal
Indlela yokucoca ikholamu ye-Centrifuge ingabhengeza i-DNA ngokukodwa ngokusebenzisa izixhobo ezikhethekileyo ze-silicon matrix adsorption, ngelixa i-RNA kunye neprotheni inokudlula kakuhle, kwaye emva koko isebenzise ityuwa ephezulu ephantsi kwe-PH ukudibanisa i-nucleic acid, ityuwa ephantsi ephezulu yexabiso le-PH yokwahlula kunye nokucoca i-nucleic acid.Iinzuzo kugxininiso oluphezulu lokuhlanjululwa, ukuzinza okuphezulu, akukho mfuneko ye-solvent ye-organic, kunye neendleko eziphantsi.Ukungalungi kukuba kufuneka ukuba i-centrifuged inyathelo ngenyathelo, amanyathelo angaphezulu okusebenza.
④ Indlela yamaso ngemagnethi
Indlela yamaso enozibuthe kukwahlula isampulu yethishu yeseli nge-lysate, ukukhulula i-nucleic acid kwisampulu, emva koko iimolekyuli ze-nucleic acid zibhengezwe ngokukodwa kumphezulu we-magnetic bead, ngelixa ubumdaka obufana neeproteni kunye neswekile bushiywe ngaphakathi. ulwelo.Ngamanyathelo okuqhekeka kwezicubu zeeseli, ukubopha i-bead magnetic nge-nucleic acid, i-nucleic acid yokuhlamba, i-nucleic acid elution, njl., i-nucleic acid ecocekileyo ifunyenwe ekugqibeleni.Iinzuzo kukusebenza okulula kunye nokusetyenziswa kwexesha elifutshane, ngaphandle kwemfuneko yesinyathelo se-centrifugation.Ineemfuno zobugcisa obuphantsi kwaye inokuqonda ukusebenza okuzenzekelayo kunye nobuninzi.Inhlanganisela ethile ye-magnetic bead kunye ne-nucleic acid yenza i-nucleic acid ekhutshiweyo kunye noxinzelelo oluphezulu kunye nococeko.Ububi kukuba ixabiso lemarike langoku liyabiza kakhulu.
⑤ Ezinye iindlela
Ukongeza kwezi ndlela zine zingentla, kukho ukuqhekeka okubilisayo, indlela yetyuwa egxininisiweyo, indlela ye-anionic detergent, indlela ye-ultrasonic kunye nendlela ye-enzymatic, njl.
Ⅲ Uhlobo lwe-nucleic acid extraction
I-Foregene ineqonga le-PCR elikhokelayo kwihlabathi, iqonga le-RNA elinezintlu ezimbini (i-DNA-kuphela + i-RNA kuphela).Iimveliso eziphambili ziquka i-DNA/RNA i-isolation kits, i-PCR kunye ne-Direct PCR reagents i-molecular lab reagents series.
① Utsalo lwe-RNA iyonke
Iisampulu ze-RNA zokukhutshwa zizonke ziquka igazi, iiseli, izicubu zezilwanyana, izityalo, iintsholongwane, njl njl. i-molecular cloning, i-Dot Blot kunye nezinye iimvavanyo.
Inxulumene ne-ForegeneI-RNA Isolation Kits
Animal Total RNA Isolation Kit--Ngokukhawuleza nangempumelelo ukukhupha ubunyulu obuphezulu kunye nomgangatho ophezulu we-RNA kwizicubu ezahlukeneyo zezilwanyana.
ISeli iyonke yeRNA Isolation Kit--Isulungeke kakhulu kwaye ikumgangatho ophezulu we-RNA iyonke inokufumaneka kwiiseli ezahlukeneyo zenkcubeko kwimizuzu eli-11.
Plant Total RNA Isolation Kit--Khipha ngokukhawuleza i-RNA esemgangathweni ophezulu kwiisampuli zezityalo ezine-polysaccharide ephantsi kunye nomxholo we-polyphenol.
IViral RNA Isolation Kit-Yahlula ngokukhawuleza kwaye uhlambulule i-RNA yentsholongwane kwiisampulu ezifana neplasma, i-serum, ulwelo lomzimba olungenaziseli kunye nezinto ezingaphaya kwamandla eeseli.
② I-Genomic DNA extraction
Iisampulu ze-Genomic DNA extraction zibandakanya umhlaba, ilindle, igazi, iiseli, izicubu zezilwanyana, izityalo, iintsholongwane, njalo njalo. -ulandelelwano lweziphumo kunye neminye imifuniselo.
Inxulumene ne-ForegeneDNA Isolation Kits
Animal Tissue DNA Isolation Kit--Ukukhutshwa ngokukhawuleza kunye nokuhlanjululwa kwe-DNA ye-genomic kwimithombo emininzi, njengezicubu zezilwanyana, iiseli, njl.
I-Blood DNA Midi Kit (1-5ml)-Ukucoca ngokukhawuleza i-DNA ye-genomic ephezulu kwigazi eli-anticoagulated (1-5ml).
Buccal Swab / FTA Card DNA Isolation Kit--Ukucoca ngokukhawuleza i-DNA ye-genomic ephezulu kwi-buccal swab / iisampuli zeKhadi le-FTA.
Plant DNA Isolation Kit-Ukucoca ngokukhawuleza kwaye ufumane i-DNA ye-genomic ephezulu kwiisampuli zezityalo (kubandakanya i-polysaccharides kunye neesampuli zezityalo ze-polyphenol)
③ Ukutsalwa kwePlasmid
I-Plasmid luhlobo lwesangqa semolekyuli encinci ye-DNA kwiiseli, engumthwali oqhelekileyo we-DNA recombination.Indlela ye-plasmid extraction kukususa i-RNA, ukwahlula i-plasmid kwi-bacterial genomic DNA, kunye nokususa iprotheni kunye nobunye ukungcola ukufumana i-plasmid ecocekileyo.
IKit yePlasmid Mini ngokubanzi-Ucoca ngokukhawuleza umgangatho ophezulu we-plasmid DNA kwiibhaktheriya eziguquliweyo zovavanyo lwebhayoloji yemolekyuli efana notshintsho kunye nokugaya i-enzyme.
④ Ezinye iintlobo zokutsalwa, ukutsalwa kwe-miRNA, njl.
Animal miRNA Isolation Kit-Ngokukhawuleza nangokufanelekileyo ukukhupha amaqhekeza amancinci e-RNA ye-20-200nt miRNA, i-siRNA, i-snRNA evela kwizicubu ezahlukeneyo zezilwanyana kunye neeseli.
Ⅳ Iimfuno ze-nucleic acid extraction kunye nesiphumo sokucocas
① Ukuqinisekisa ingqibelelo yesakhiwo sokuqala se-nucleic acid.
② Ukunciphisa ukuphazamiseka kweeprotheni, iswekile, i-lipids kunye nezinye ii-macromolecules
③ Akufuneki kubekho i-solvent ye-organic okanye i-concentration ephezulu yee-ion zetsimbi ezinokuthintela i-enzyme kwiisampuli ze-nucleic acid.
④ I-RNA kunye nolunye ungcoliseko lwe-nucleic acid kufuneka lupheliswe xa ukhupha i-DNA, kwaye ngokufanayo.
Ixesha lokuposa: Nov-24-2022
