Gutandukanya ADN kuva E. Coli nuburyo bwibanze muri ibinyabuzima bya molecular. Iyi ngingo izagutwara muburyo bwose, itanga intambwe zirambuye hamwe nibisobanuro birambuye, kugenzura usobanukiwe siyanse hamwe nibice bifatika byuburyo. Waba umushakashatsi wibanze cyangwa mushya kuri laboratoire, iki gitabo kizaba umutungo wingenzi.
Imyiteguro yo guhagarikwa
● Icyegeranyo cya E. Coli selile
Intambwe yambere mugutandukanya ADN kuva E. Coli ikubiyemo gukusanya selile za bagiteri. Ibi mubisanzwe bisaba gukura E. Coli muburyo bukwiye kugeza aho igeze ku muvuduko wa logarithmic. Igihe ni ngombwa kuko selile muriki cyiciro ni ibintu byinshi kandi byoroshye kuri LYSE, bizavamo umusaruro wo hejuru wa ADN.
● Gukomeza selile muri buffer ikwiye
Yakusanyije ingirabuzimafatizo noneho yasukuwe muri buffer ibereye. Ihitamo rusange ni tris - edta (te) buffer, ifasha gukomeza ubusugire bwa ADN mugihe cyo gukuramo. Buffer akora intego nyinshi: ihatira PH, Chelate yamaguru ya Tariki ya DNA ishobora gutesha agaciro ADN, kandi itanga ibidukikije bya Iomical kubitekerezo byakurikiyeho.
Centrifugation kuri selile
● Ibipimo bya centrifugation (umuvuduko nigihe)
Nyuma yo guhaga selile, guhagarikwa gukorerwa centrifugation kuri pellet. Umuvuduko ukabije nigihe ni ibipimo bikomeye. Mubisanzwe, centrifugation ikorwa ahagana hafi 4000 - 6.000 G kuri 10 - Iminota 15 kuri 4 ° C. Ibi byemeza ko selile ikora pellet hepfo yumuyoboro wa Centrifuge.
● Akamaro ko gutondekanya neza
Pelleting ikwiye ni ngombwa kugirango utandukanye selile ziva mu mico yo hagati nibindi bice bifatika. Ibyiza - byashizeho pellet bituma habaho intambwe yakurikiyeho kandi neza, kugirango ubuze selile kandi rero, bityo, umusaruro wa ADN.
Gukuraho Ikizere
Tekinike yo gukuraho ndengali
Ingirabuzimafatizo zimaze guterwa, ndengakanga (amazi hejuru ya pellet) agomba gukurwa neza atabangamiye pellet. Ibi mubisanzwe bikorwa ukoresheje micropipette. Ni ngombwa gukora iyi ntambwe neza kugirango wirinde gutakaza selile.
● Guhata igihombo gito cya pellet
Kugirango itakaza igihombo gito kuri pellet selile ikubiyemo gukoreshwa neza kandi, nibiba ngombwa, ibyiciro byinshi byibice no gukuraho ndengakaga. Intego ni ugukomeza selile nyinshi zishoboka murutonde rwa ADN ntarengwa ya ADN.
Ongeraho igisubizo cya Nuclei Lysis
● Ibigize Nuclei LILSIS
Igisubizo cya Nucleilis gisanzwe kirimo ibikoresho (nka SDS), buffer (nka Triss - HCL), hamwe numukozi wa CCLe (nka EDTA). Ibikoresho byo gufatanya guhagarika imiyoboro ya selile n'ibahasha ya kirimbuzi, irekura ibiri muri selile, harimo na ADN, mu gisubizo.
Uruhare rwo kumena urukuta rw'akagari
Igisubizo cya Nuclei ntabwo vese gusa gusa lymbrane gusa, ahubwo nahanagura poroteyine na lipide, bamenagura neza inkuta zakagari nibahasha ya kirimbuzi kugirango barekure ADN mubisubizo.
Gusundukira selile
● Wice woroheje kugirango wirinde kogosha ADN
Iyo igisubizo cya Nuclei kimaze kongerwaho, selile zigomba gusubukura witonze kugirango wirinde kogosha ADN. Gusinzira birashobora kumena ADN mubice bito, bishobora kuba ikibazo kubisabwa kumanuka bisaba hejuru - molecular - ADNO ADN.
● Komeza guturuka
Gusubukira byuzuye bireba ko selile zose zinyeganyega kimwe, zikangura ADN. Ibi birashobora kugerwaho nubwitonzi cyangwa gutobora igisubizo kumuvuduko muto.
Incubation kuri lyse selile
Igenamiterere ry'ubushyuhe kuri incubation
Ingirabuzimafatizo zisukuye ziterwa nubushyuhe bwihariye kugirango umenye neza lysis. Ibi mubisanzwe bikozwe muri 37 ° C kugeza 55 ° C. Ubushyuhe nyabwo bumara buratandukanye bitewe na protocole nibisabwa byihariye byimiti ya ADN ikoreshwa.
● Igihe gisabwa kuri lysis nziza
Igihe gisanzwe kuri incubation kiri hagati yiminota 20 kugeza 30, ariko irashobora guhinduka ukurikije imikorere ya selile. Incuvation yigihe kirekire irashobora gukenerwa kuri lysis yuzuye ariko igomba gushyira mu gaciro ingaruka zo gutesha agaciro dna.
Gukonjesha ubushyuhe bwicyumba
● Akamaro ko gukonjesha buhoro buhoro
Nyuma yo guhagarika incukobation, Lysate irakingurutse buhoro buhoro ubushyuhe bwicyumba. Gukonjesha buhoro buhoro bifasha muguterana na ADN kandi kugabanya ibyago byo guhagarika umutima, bishobora guteza ADN.
Ingaruka kuri ADN na Debrist
Gukonjesha ubushyuhe bwicyumba butuma imyanda ya selile iciriritse, yorohereza gutandukanya ADN mugihe cyakurikiyeho. Ibi kandi bifasha gushimangira ibikorwa bya enzyme no korohereza kuvanaho RNA na RNESE.
Ongeraho igisubizo cya RNASE
● intego ya rnase muburyo
RNASE Rnase guhitamo indogobe rna, hasigara ya ADN itameze neza.
Kubuza RNA kwanduza
Kurinda kwanduza RNA ningirakamaro kubisabwa bisaba ADN yera, nka PCR na SCIED. Guhangana knase bituma ADN yitaruye itarangwamo RNA.
Isuku rya ADN
● Uburyo bwo gutandukanya ADN uhereye kubice bya selile
Uburyo bwinshi burashobora gukoreshwa muguhanagura ADN uhereye kumasaha. Ibi birimo phenol - Gukuramo chloroform, imvura ya ethanol, no gukoresha ubucuruzi bwa E. Coli DNA. Buri buryo bufite ibyiza kandi ibibi, hamwe nibikoresho byubucuruzi bitanga byoroshye kandi bihamye ibisubizo.
Gutekereza kwa ADN
Isuku nikintu gikomeye kugirango atsinde ibicuruzwa byanyuma. Ibikoresho byubucuruzi, nka kuvura selire e. Coli Dna Kit, yashizweho kugirango umusaruro usumba - Isuku ikuraho neza poroteyine, lipids, hamwe nabandi banduye.
Ububiko no Gukemura ADN Yigunze
● Imyitozo myiza yo kubika ADN
Igihe kimwe cyezwa, ADN igomba kubikwa muri buffer ibereye, nka teffer, kuri - 20 ° C cyangwa - 80 ° C kurera kuva kera - Ububiko. Irinde Freeze kenshi - Thaw inzinguzingo uko zishobora gutezimbere kwa DNA.
Kubungabunga ADN ubunyangamugayo bwa ADN kubisabwa kumanuka
Kubungabunga ubunyangamugayo bwa DNN, koresha sterile, nuclease - igituba cyibisubizo. Ibi byemeza ko ADN ikomeje kutatandurwa kandi ikwiriye gusaba nka cloni, ikurikirana, na PCR.
HafiBluekit
Jiamssu Hillgene yashyizeho icyicaro cyayo (10,000㎡ Ikigo cya GMP na R & D) i Suzhou, na Sunzhen na Shanghan na Shanghan na Shanghai, bagura urusobe rwarwo mugihugu hose. Urubuga rwa karolina y'Amajyaruguru muri Amerika ubu rurimo kubakwa, gukomeza gukwirakwiza isi yose. Hillgene yubatse inzira nyabagendwa mugutezimbere ibicuruzwa bya selile, kuvavumburwa no gukora ibikorwa bya acide ya nucleic, imitekerereze yubusa, iterambere ryimigambi rifunze, na QC bigerageza. Ibicuruzwa by'Ubururu byateguwe kugira ngo bigenzurwe neza, birebe ko udushya turimo imivuruke.
Igihe cyagenwe: 2024 - 09 - 05 14 14:47:03
