Ukuhlukanisa i-DNA kusuka e-E. Coli kuyinqubo eyisisekelo ku-Bilefanelar Biology. Lo mbhalo uzokuhamba kuyo yonke inqubo, ukuhlinzeka ngezinyathelo ezinemininingwane nezincazelo, ukuqinisekisa ukuthi uyakuqonda zombili iSayensi kanye nezinto ezisebenzayo zenqubo. Noma ngabe ungumcwaningi onamava noma omusha welebhu, lo mhlahlandlela uzoba insiza ebalulekile.
Ukulungiselela ukumiswa kweseli
● Ukuqoqwa kwamaseli we-E. Coli
Isinyathelo sokuqala ehlukanisa i-DNA kusuka ku-E. Coli kubandakanya ukuqoqa amaseli amagciwane. Lokhu kuvame ukudinga ukukhula kwe-E. Coli ngendlela efanelekile ye-liquid kuze kube yilapho ifinyelela esigabeni sokukhula kwe-logarithmic. Isikhathi sibalulekile ngoba amaseli kulesi sigaba asebenza kakhulu futhi kulula kakhulu ukuzulazula ngokuthola isivuno esiphakeme se-DNA.
● Ukuvuselela amaseli ku-buffer efanele
Amaseli aqoqiwe bese avuselelwa kwi-buffer efanelekile. Ukhetho olujwayelekile luyi-tris - i-edta (te) buffer, esisiza ukugcina ubuqotho be-DNA ngesikhathi senqubo yokukhishwa. I-Buffer isebenza ngezinhloso eziningi: Iqinisa i-PH, ifuna ukuhlukaniswa kwezindawo ezihlukile ezingalimaza i-DNA, futhi zinikeze indawo efanelekile ye-ionic yokuphendula okulandelayo kwe-enzymatic.
I-Centrifugation kuma-pellet cell
● Amapharamitha we-centrifugation (isivinini nesikhathi)
Ngemuva kokuvuselela amaseli, ukumiswa kufakwa ngaphansi kwe-Centrifugation ukuze ugobe amaseli. Ijubane le-centrifugation nesikhathi amapharamitha abucayi. Imvamisa, i-centrifugation yenziwa cishe ngo-4,000 - 6,000 g for 10 - Imizuzu engu-15 ku-4 ° C. Lokhu kuqinisekisa ukuthi amaseli akha i-pellet eqinile ezansi kwe-tube ye-centrifuge.
● Ukubaluleka kwe-pelleting efanele
I-pelleting efanelekile ibalulekile ukuze uhlukanise amaseli kusuka ekukhuleni kwaphakathi nezinye izingxenye ezi-soluble. I-pellet enhle - eyenziwe kahle yenza izinyathelo ezilandelayo zibe lula futhi zisebenze kahle, ziqinisekise ukulahleka okuncane kwamaseli futhi, ngakho-ke, isivuno esikhulu se-DNA.
Ukususwa kwe-supernatant
● Amasu wokususwa kwe-supernatant
Lapho amaseli esephepheni, i-supernatation (uketshezi olungenhla kwe-pellet) kumele lisuswe ngokucophelela ngaphandle kokuphazamisa i-pellet cell. Lokhu kuvame ukwenziwa kusetshenziswa i-micropipette. Kubalulekile ukwenza lesi sinyathelo ngokucophelela ukugwema ukulahlekelwa amanye amaseli.
● Ukuqinisekisa ukulahleka okuncane kwe-cellet cellet
Ukuqinisekisa ukulahleka okuncane kwe-pellet cell kufaka phakathi i-Pipetting ngokucophelela futhi, uma kunesidingo, imijikelezo eminingi ye-centrifugation kanye nokususwa okuphezulu. Umgomo ukugcina amaseli amaningi ngangokunokwenzeka kwi-pellet ukuze uthole ukululama okukhulu kwe-DNA.
Ukungezelelwa kwesixazululo se-nuclei lysis
● Izingxenye zesisombululo se-nuclei lysis
Isixazululo se-nuclei lysis ngokuvamile siqukethe i-detergent (njenge-SDS), i-buffer (efana ne-tris - hcl), ne-ejenti ye-chelating (njenge-Edta). I-deteggent iphazamisa ulwelwesi lwamaseli nemvilophu yenuzi, ukukhulula okuqukethwe kwamaselula, kufaka phakathi i-DNA, kusisombululo.
● Indima yokwephula izindonga zeseli
Isixazululo se-nuclei lysis asigcini nje ngokunwebeka ulwelwesi lweseli kuphela kodwa futhi sibonisa amaprotheni nama-lipids, ngokubhidliza izindonga zeseli kanye nezimvilophu zenuzi ukukhulula i-DNA kwikhambi.
Ukuvuselelwa kwamaseli
● Pipetting emnene ukugwema ukufuya kwe-DNA
Lapho sengezwa isixazululo se-nuclei lysiss, amaseli adinga ukuvulwa kabusha ngobumnene ukugwema ukucheba kwe-DNA. Ukugunda kungaphula i-DNA ibe yizicucu ezincane, ezingaba yinkinga ngezicelo ezaphansi ezidinga ukuphakama - ama-molecular - Isisindo se-DNA.
● Ukuqinisekisa ukuvuselelwa okuphelele
Ukuvuselelwa okuphelele kuqinisekisa ukuthi wonke amaseli anwetshwa ngokufana, akhulisa ukululama kwe-DNA. Lokhu kungatholakala nge-Pipetting emnene noma i-vortexing ikhambi ngesivinini esiphansi.
Ukufakwa kumaseli we-lyse
● Izilungiselelo zokushisa zokufakwa ngaphakathi
Amaseli avuselelwa kabusha afakwa emazingeni okushisa athile ukuze aqinisekise i-lysis ephelele. Lokhu kuvame ukwenziwa ku-37 ° C kuye ku-55 ° C. Ukushisa okuqondile nobude besikhathi kungahluka ngokuya nge-protocol kanye nezidingo ezithile zekhithi ye-DNA Ukuhlukaniswa.
● Isikhathi esidingekayo ukuze i-lysis esebenzayo
Isikhathi esijwayelekile sokufakwa kokufunga siphakathi kwemizuzu engama-20 kuye kwangama-30, kepha singashintshwa ngokususelwa ekusebenzeni kahle kwe-cell shiys ebonwa. Ukufakwa isikhathi eside kungadingeka ukuze ku-lysis ephelele kepha kufanele kube nokulinganisela ngokumelene nengozi yokuwohloka kwe-DNA.
Ukupholisa kokushisa kwegumbi
● Ukubaluleka kokupholisa kancane kancane
Ngemuva kokufakwa ngaphakathi, i-lysate kancane inyuswe kancane ekushiseni kwegumbi. Ukupholisa kancane kancane kusiza ekuqiniseni i-DNA futhi kunciphise ubungozi bokushaqeka okushisayo, okungenza kahle i-DNA.
● Imiphumela ku-DNA ne-Cellular Debris
Ukupholisa ekushiseni kwegumbi kuvumela imfuthwane yamaselula ukucacisa, okwenza kube lula ukuhlukanisa i-DNA ngezinyathelo ezalandela. Lokhu kusiza nokuqinisa imisebenzi ye-enzyme futhi kusiza ukususwa kwe-RNA ngokwelashwa kweRnase.
Ukungezelelwa kwesixazululo se-rnase
● inhloso ye-rnase kwinqubo
Isixazululo se-RNASE sengezwa ku-defader RNA, okungenzeka ukuthi sihlanze nge-DNA futhi siphazamise ngezicelo ezaphansi. I-RNASE ekhetha ukugaya i-RNA, ishiya i-DNA ingenangqondo.
● Ukuvimbela ukungcoliswa kwe-RNA
Ukuvimbela ukungcoliswa kwe-RNA kubalulekile ukuthi izinhlelo zokusebenza ezidinga i-DRA ehlanzekile, njenge-PCR nokulandelana. Ukwelashwa kwe-RNASE kuqinisekisa ukuthi i-DNA ekhethekile ikhululekile ekungcolisweni kwe-RNA.
Ukuhlanzwa kwe-DNA
● Izindlela zokuhlukanisa i-DNA ezivela kwezinye izingxenye zamaselula
Izindlela eziningana zingasetshenziswa ukuze zihlanze i-DNA kusuka eLysate. Lokhu kufaka phakathi i-phenol - ukukhishwa kwe-chlorform, i-ethanol yezulu, nokusebenzisa ezentengiso E. Coli DNA KINITS. Indlela ngayinye inezinhle nezinzuzo zayo, ngamakhithi okuhweba ahlinzeka lula kanye nemiphumela engaguquki.
● Ukucatshangelwa kokuhlanzeka kwe-DNA
Ubumsulwa buyisici esibucayi sempumelelo yezicelo ezaphansi. Ama-Kits ezentengiso, njengokwelashwa kweseli e. Coli DNA Kit, aklanyelwe ukuveza ukuphakama - I-DNA purness ngokususa ngempumelelo amaprotheni, ama-lipid, nezinye izinto ezingcolile.
Isitoreji nokuphathwa kwe-DNA Ebolated
● Imikhuba emihle kakhulu yokugcina i-DNA
Uma sekuhlanzwa, i-DNA kufanele igcinwe kwi-buffer efanelekile, njenge-teuffer, ku- - 20 ° C noma - 80 ° C isikhathi eside - ukugcinwa kwethemu. Gwema iqhwa kaningi - Imijikelezo emincane njengoba ingadala ukonakala kwe-DNA.
● Ukugcina ubuqotho be-DNA ngezinhlelo zokusebenza ezaphansi
Ukuze ulondoloze ubuqotho be-DNA, sebenzisa oyinyumba, nuclease - amashubhu wamahhala nezisombululo. Lokhu kuqinisekisa ukuthi i-DNA ihlala ingcolile futhi ilungele izinhlelo zokusebenza ezifana nokulandelana, ukuhlela, ne-PCR.
MaqondanaI-Bluekit
UJiangsu Hillgene wasungula indlunkulu yawo (izitshalo ezingama-10,000 000 GMP kanye ne-R & D Contrect) eSuzhoud, eSuzhou, idolobha laseLakeshore lechibi elihle laseTaihu, kanye neziza ezimbili zokukhiqiza eShenzhen naseShanghai, zidlulisela inethiwekhi yayo yokukhiqiza ezweni lonke. Isayithi laseNorth Carolina e-US njengamanje lakhiwa, lisakazeka futhi ukuba khona kwalo komhlaba wonke. IHillgene yakhele indlela ebonakalayo yokwakhiwa kwemikhiqizo yokwelashwa kwamaselula, ukusuka ekutholweni kuya ekulethweni, ngamapulatifomu okukhiqiza i-nucleic acid, i-serum - ukumiswa kwamahhala kwesiko, ukuthuthukiswa kwenqubo okuvaliwe, kanye nokuhlolwa kwe-QC. Imikhiqizo ye-Bluekit yenzelwe ukulawulwa kwekhwalithi, ukuqinisekisa ukuphumelela kwamaselula wezokwelapha amaselula.
Isikhathi Seposi: 2024 - 09 - 05 14:47:03
