Ukuzahlula iDNA evela e-E. Coli yinkqubo esisiseko ye-molecular. Eli nqaku liza kukuhamba kuyo yonke le nkqubo, ukubonelela ngamanyathelo aneenkcukacha kunye neenkcazo, ukuqinisekisa ukuba uyasiqonda isayensi kunye nemiba esebenzayo yenkqubo. Enoba ungumthengisi onamava okanye umtsha-mfutshane kwilebhu, esi sikhokelo siya kuba sisibonelelo esixabisekileyo.
Ukulungiswa kokumiswa kweseli
● Ukuqokelelwa kweeseli ze-E. Coli
Inyathelo lokuqala lokuzahlula iDNA evela e-E. Coli ibandakanya ukuqokelela iiseli zentsholongwane. Oku kudla ngokufuna ukukhula e. I-Coli kwi-soli enesigaba esifanelekileyo de ifike kwinqanaba lokukhula kwe-logo. Ixesha libalulekile kuba iiseli kweli nqanaba zisebenza kakuhle kwaye zilula kwiLise, eziya kukhokelela kwi-DNA ephezulu.
● Iiseli zokuhambisa kwakhona kwi-buffer efanelekileyo
Iiseli eziqokelelweyo zenziwe ngelo xesha ziphindaphindeke kwi-buffer efanelekileyo. Ukhetho oluqhelekileyo yi-Tris - I-EDTA (TE) Buffer, enceda ukugcina ingqibelelo yeDNA ngexesha lenkqubo yokukhuphela. I-buffer isebenzele iinjongo ezininzi: Ukuzinzisa i-PH, i-chelates yeendlela ezinokuthi zingade kuhlehlise iDNA, kwaye ibonelela ngemozulu ye-ionic yokuphendula emva kwexesha.
I-Centrifugation kwiiseli ze-Pellet
● Iiparameter ze-centrifugation (isantya kunye nexesha)
Emva kokubuya ngokusasaza iiseli, ukumiswa kufakwe kwisentimitha ukuya e-pellet iiseli. Isantya sokwakhiwa kunye nexesha ziiparamitha ezibalulekileyo. Ngokwesiqhelo, ukwenziwa kwekhulu lwenziwa malunga ne-4,000 - I-6,000 g nge-10 - Imizuzu eli-4 nge-4 ° C. Oku kuqinisekisa ukuba iiseli zenza i-pellet eqinileyo ezantsi kwe-centrifuge tube.
● Ukubaluleka Kokubamba okufanelekileyo
I-Pellet efanelekileyo ibalulekile ukwahlula iiseli ezivela kwiziko eliphakathi kunye nezinye izinto ezinyibilikayo. I-Pellet ye-Pellet yenza ukuba amanyathelo alandelayo alandelayo kwaye asebenza ngokufanelekileyo, aqinisekisa ukulahleka okuncinci kweeseli kwaye, ngenxa yoko, ubuninzi be-DNA.
Ukususwa kwe-supernatatant
● Iindlela zokususwa kwe-Superntual
Nje ukuba iiseli zifakwe, i-supernatant (i-windows elingaphezulu kwe-pellet) kufuneka isuswe ngononophelo ngaphandle kokuphazamisa ipellet yeseli. Oku kuhlala kwenziwe kusetyenziswa i-micropipette. Kubalulekile ukwenza eli nyathelo licwangcise kakuhle ukunqanda ukuphulukana naziphi na iiseli.
● Ukuqinisekisa ukulahleka okuncinci kwepellet yeseli
Ukuqinisekisa ukulahleka okuncinci kwe-pellet yeseli kubandakanya ukuqubudiza ngononophelo kwaye, ukuba kukho imfuneko, imijikelezo emininzi ye-centrifugation kunye nokususwa okungaphezulu. Injongo kukugcina iiseli ezininzi kangangoko kunokwenzeka kwi-pellet ye-DNA ephezulu.
Ukongezwa kwe-nuclei lysis
● Izinto ze-nuclei lysis
I-nuclei lysis yesisombululo iqulethe isicoci (njenge-SDS), i-buffer (njenge-tris - i-HCL), iarhente yokutshekisha (njenge-Edta). Isithintelo esiphazamisa imvulophu yeseli nenyukliya, ukukhulula imixholo yeselula, kubandakanya iDNA, kwisisombululo.
● Indima yokwaphula iindonga zeseli
Isisombululo se-nuclei lysis kungekuphela nje kwe-membrane yeseli kodwa ikwakhanyele iiprotein kunye nee-lipids, ngokuphumelelayo iindonga zeseli kunye nemvulophu yenyukliya yokukhupha i-DNA kwisisombululo.
UKHUSELEKO LWEZIPHUMO
● Ipatteyitha ethambileyo ukuthintela ukucheba kwe-DNA
Nje ukuba i-nuclei lysis iyongezwa, iiseli kufuneka ziphinde ziphinde zibe ngobunono ukuze ziphephe ukucheba kwe-DNA. Ukucheba kunokwaphula i-DNA ibe ngamaqhekeza amancinci, enokuba yingxaki kwizicelo ezihamba ezantsi ezifuna ukuphezulu - I-molecular - I-DNA ye-DNA.
● Ukuqinisekisa ukuphinda-phinda
Ukuphinda uqhubeke nokuphinda uqhubeke kuqinisekisa ukuba zonke iiseli ziyinto efanayo, yandisa i-DNA. Oku kunokufezekiswa ngumjelo othambileyo okanye ukudubula isisombululo kwisantya esisezantsi.
I-alkowation kwiiseli ze-lyse
● Useto lobushushu lokufaka
Iiseli ezivunyiweyo zifakwe kubushushu obuthile ukuqinisekisa ukuba i-Lysis epheleleyo. Oku kuhlala kwenziwe nge-37 ° C ukuya kwi-55 ° C. Olona qondo lobushushu ngqo kunye nexesha linokwahluka kuxhomekeka kwiprotocol kunye neemfuno ezithile zekhithi ye-DNA yokusetyenziswa.
● Ixesha elifunekayo kwi-lysis esebenzayo
Ixesha elifanelekileyo lokufakwa phakathi kwama-20 ukuya kwi-30 imizuzu, kodwa inokuhlengahlengiswa ngokusekwe kwi-Lyfisis yeseli ibonwe. Ubume bexesha elide bunokuba yimfuneko kwi-Lysis epheleleyo kodwa kufuneka ilungelelaniswe ngokuchasene nomngcipheko we-DNA Deradition.
Ukupholisa kubushushu begumbi
● Ukubaluleka kokuphola kancinci
Emva kokufakwa, i-lyssate ithola ngokuthe ngcembe kubushushu begumbi. Ukupholisa ngokuthe ngcembe kuyanceda ukuzinzisa i-DNA kwaye kuncitshiswe umngcipheko wokothuka, onokuthi angayiniki da iDNA.
● Iziphumo kwi-DNA kunye ne-sellum yeselula
Ukupholisa kubushushu begumbi kuvumela inkunkuma iselula ukuya kuthintela, yenze kube lula ukwahlula i-DNA kumanyuko alandelayo. Oku kukwanceda ukuzinzisa imisebenzi ye-enzyme kwaye iqhubele phambili ekususweni kweRNA ngonyango lwe-rnase.
Ukongezwa kwesisombululo se-rnase
● Injongo ye-rnase kwinkqubo
Isisombululo se-rnase songezwa kwi-Degrade RNA, enokuhlambulula i-CO - Ukuhlambulula nge-DNA kwaye uphazamise izicelo ezisezantsi. I-Rnise ngokukhethekileyo i-Diackers i-RNA, ishiya i-DNA iqaqanjelwe.
● Ukuthintela ungcoliseko lwe-rna
Ukuthintela ungcoliseko lwe-RNA kubalulekile kwizicelo ezifuna i-DNA ecocekileyo, ezinjenge-PCR kunye nendlela yokulandelelana. Unyango lwe-rnase luqinisekisa ukuba i-DNA ekwanti ikhululekile kwi-RNA ingamanyala.
Ukuhlanjululwa kweDNA
● Iindlela zokwenza ukwahlula iDNA kwezinye izinto zeselula
Iindlela ezininzi zinokusetyenziselwa ukucoca i-DNA kwiLissate. Oku kubandakanya i-phenol - i-chlorofor yokutyunjwa, kunye nokusebenzisa intengiso e. Coli DNA Kits. Indlela nganye inezinto zayo kwaye inobuchule, kunye neeKits zorhwebo ezibonelela kakuhle neziphumo ezingaguqukiyo.
● Ukuqwalaselwa kokucoceka kwe-da
Ukucoceka yinto ebaluleke kakhulu kwimpumelelo yezicelo ezihla ezantsi. Iikhithi zorhwebo, ezinje ngeseli e-enrapy E. I-Coli DNA Kikhi, yenzelwe ukuvelisa phezulu - Ukucoceka kwe-DNA ngokususa iiprotein, i-lipids, kunye nezinye izinto ezingcolisayo.
Ukugcina kunye nokuphatha i-DNA ekwanti
● Iindlela ezilungileyo zokugcina iDNA
Nje ukuba ihlanjululwe kwi-buffer efanelekileyo, njenge-buffer efanelekileyo, kwi - 20 ° C okanye - i-80 ° C ixesha elide - ukugcinwa kwexesha elide. Kunqande ukukhululeka rhoqo - Imijikelezo ye-thaw njengoko inokubangela ukonakaliswa kwe-DNA.
● Ukugcina ukuthembeka kwe-DNA kwizicelo ezisezantsi
Ukugcina ingqibelelo yeDNA, sebenzisa i-nucle, iityhubhu zasimahla kunye nezisombululo. Oku kuqinisekisa ukuba iDNA ihlala ingalunganga kwaye ilungele izicelo ezinje ngokujikeleza, ukulandelelana, kunye ne-pcc.
MalungaI-Bluekit
UJiangsu Hillgene wamisela ikomkhulu layo (i-10,000 gesela i-GMP) eSuzhou, kwindawo ye-Wozhong, kunye neSuzhou, i-suzhou, i-sunghoi, i-shanghai, i-Shanghai, eyandisa uthungelwano lwemveliso kwilizwe liphela. Indawo ye-North Carolina e-US iqala ukwakhiwa, ngakumbi ukusasaza ubukho bayo behlabathi. Hillgene has built an express pathway for developing cellular therapy products, from discovery to delivery, with platforms for nucleic acid manufacturing, serum-free suspension culturing, closed process development, and QC testing. Iimveliso zeBluekit zenzelwe ulawulo lomgangatho, ukuqinisekisa impumelelo yokuphumelela kweselfowuni.
IXESHA LOKUQALA: 2024 - 09 - 05 14:47:03
