I-EndoFree Plasmid Maxi Kit

Izimo zokugcina

I-RNaseA kufanele igcinwe ku -30 ~ -15℃ futhi ihanjiswe ku-≤0℃.

I-Endotoxin Scavenger ingagcinwa ku-2 ~ 8℃ inyanga eyodwa, igcinwe ku--30 ~ -15 ℃ ukuze igcinwe isikhathi esidefuthi ithuthwa ngo-≤0℃.

Ezinye izingxenye kufanele zigcinwe ekamelweni lokushisa (15 ~ 25 ℃) futhi zihanjiswe ekamelweni lokushisa.

Izingxenye

RNaseA:10 mg/ml, esetshenziselwa ukususa i-RNA.

Isithameli se-P1:ibhafa yokumiswa kwebhaktheriya, engeza i-RNaseA ku-Buffer P1 ngaphambi kokusetshenziswa kokuqala.

Isivimbeli se-P2:i-bacterial lysis buffer (equkethe i-SDS/NaOH).

Isithameli se-P4:i-neutralize buffer.

I-Endotoxin Scavenger:khipha ngempumelelo i-endotoxin ekukhishweni kwe-plasmid engahluziwe.

I-Buffer PW:geza isigcinalwazi, engeza umthamo oshiwo we-ethanol ngaphambi kokuwusebenzisa okokuqala.

I-Buffer TB:i-lution buffer.

I-FastPure DNA Maxi Columns:i-plasmid DNA adsorption columns.

Amashubhu eqoqo 50 ml:filtrate iqoqo amashubhu.

I-Endotoxin-free Collection Tube:amashubhu okuqoqwa kwe-plasmid DNA.

Izinto Ezilungisiwe

I-ethanol ephelele, isopropanol, 50 ml amashubhu e-centrifuge ayindilinga aphansi kanye nama-endotoxin angama-50 mlamashubhu centrifuge.

Izinhlelo zokusebenza

Lo mkhiqizo ulungele ukukhishwa okukhulu kwama-plasmids kusuka ku-150 - 300 ml yesisombululo sebhaktheriya.isiko ubusuku bonke.

Inqubo Yokuhlola

1. Thatha u-150 – 200 ml (ongekho ngaphezu kuka-300 ml) wesisombululo sebhaktheriya esikhuliswe ubusuku bonke kanye ne-centrifugemayelana ne-11,000 rpm (12,000 × g) ye-1 - 2 min.Lahla amandla amakhulu futhi uqoqe amagciwane.

Δ Lapho uqoqa ngaphezu kuka-50 ml wesisombululo sebhaktheriya, amagciwane angaqoqwa ngokwengeza isisombululo sebhaktheriya, i-centrifugation, ukulahla amandla amakhulu nezinye izinyathelo kushubhu efanayo engu-50 ml

izikhathi eziningi.

2. Engeza u-7.5 ml we-Buffer P1 (sicela uhlole ukuthi i-RNaseA yengeziwe ku-Buffer P1) ku-centrifugeithubhu eliqukethe amagciwane futhi lixube kahle nge-vortex noma ngepayipi.

Δ Ukumiswa kabusha okuphelele kwamabhaktheriya kulesi sinyathelo kubalulekile ekukhiqizeni, futhi akufanele kube khona izigaxa zebhaktheriya ngemva kokumiswa kabusha.Uma kukhona ama-bacterial clumps angaxutshwe kahle, azothinta i-lysis, okuholela ekuvuneni okuphansi nokuhlanzeka.Uma i-OD600 yesisombululo sebhaktheriya ingu-0.65, kutuswa ukuthi u-7.5 ml we-Buffer P1 usetshenziswe uma ukhishwa ku-150 ml wesisombululo sebhaktheriya;uma i-OD600 ingu-0.75, kufanele kusetshenziswe u-8 ml we-Buffer P1 kanye nevolumu ye-Buffers P2 ne-P4 kufanele ishintshwe ngokufanele.Uma umthamo wesisombululo sebhaktheriya ukhuphuka ube ngu-200 ml, kunconywa ukuthiivolumu ye-Buffers P1, P2, ne-P4 inyuswe ngokulinganayo.

3. Engeza u-7.5 ml we-Buffer P2 ekumisweni kwebhaktheriya kusukela esinyathelweni sesi-2 bese uhlanganise ngobumnene phezulu naphansi u-6 - 8.izikhathi futhi ufukamele ekamelweni lokushisa 4 - 5 min.

Δ Guqula ngobumnene ukuze uxube kahle.I-Vortexing izobangela ukuhlukana kwe-genomic DNA, okuholela ezincekwini ze-genomic DNA ku-plasmid ekhishiwe.Ngalesi sikhathi, ikhambi liba ne-viscous futhi liguquguquke, libonisa ukuthi amabhaktheriya ase-lysed ngokugcwele.Ubude besikhathi akufanele budlule imizuzu emi-5 ukugwema ukucekelwa phansi kwama-plasmids.Uma ikhambi lingacacile, kungase kube namabhaktheriya amaningi kakhulu abangelai-lysis engaphelele, ngakho-ke inani lamabhaktheriya kufanele lincishiswe ngokufanele.

4. Engeza u-7.5 ml we-Buffer P4 ekumisweni kwebhaktheriya kusukela esinyathelweni sesi-3 futhi uguqule ngokushesha izikhathi ezingu-6 - 8 ukuze uvumele ikhambi ukuthi linciphise ngokuphelele i-Buffer P2.Ngalesi sikhathi, i-white flocculent precipitate kufanele ivele.I-Centrifuge cishe ngaphezu kuka-11,000 rpm (12,000 × g) imizuzu eyi-10 – 15, faka ngokucophelela i-supernatant eshubhuni elisha eliyindilinga eliphansi elingu-50 ml (elizilungiselele), futhi ugwemelangazelela imvula emhlophe entantayo.

Δ Engeza i-Buffer P4 bese uguqule ngokushesha ukuze uxube kahle.Shiya ishubhu lime kuze kube yilapho imvula emhlophe isatshalaliswa ngokulinganayo kuso sonke isisombululo ukuze uvimbele ukukhiqizwa kwemvula yendawo okungase kuthinte ukungathathi hlangothi.Uma ingekho i-uniform white flocculent precipitate ngaphambi kwe-centrifugation futhi i-supernatant ingacacile ngemuva kwe-centrifugation, ishubhu ingabai-centrifuged enye imizuzu emi-5.

5. Engeza izikhathi ezingu-0. 1 ivolumu (u-10% wevolumu ephezulu, cishe u-2.2 ml) we-Endotoxin Scavenger ku-supernatant usuka esinyathelweni sesi-4 bese uguqule ukuze uxube.Beka isixazululo endaweni yokugezela yeqhwa noma ufake eqhweni elichotshoziwe (noma efrijini lesiqandisi) imizuzu emi-5 kuze kube yilapho isixazululo sishintsha sisuka eqhweni siye kucace futhi sobala (noma sisamile.i-turbid kancane), futhi ngezinye izikhathi uxube izikhathi eziningana.

Δ Ngemuva kokuthi i-Endotoxin Scavenger yengezwe kunamandla angaphezu kwavamile, i-supernatant izoba nesiphithiphithi kodwai-supernatant kufanele icace (noma ibe yimfucumfucu kancane) ngemva kokupholisa okugeza eqhweni.

6. Ngemva kokuba i-supernatant ibekwe ekamelweni lokushisa (>25℃) imizuzu engu-10 – 15, izoba nesiphithiphithi njengobaizinga lokushisa layo liyakhuphuka lifinyelele izinga lokushisa lekamelo.Khona-ke i-supernatant kufanele iguqulwe ukuze ixube.

Δ Uma izinga lokushisa legumbi liphansi noma ufuna ukunciphisa isikhathi sokukhipha, i-supernatant ingafukanyelwa endaweni yokugeza yamanzi engu-37 ~ 42 ℃ imizuzu engu-5 - 10 futhi isinyathelo esilandelayo singenziwa ngemva kwe-supernatant.iba nesiphithiphithi.

7. Beka i-supernatant cishe ku-11,000 rpm (12,000 × g) imizuzu engu-10 ekamelweni lokushisa (izinga lokushisa kufanele libe>>25℃) ukuze uhlukanise isigaba.Isigaba se-aqueous esiphezulu siqukethe i-DNA kuyilapho ungqimba oluphansi lwesigaba samafutha aluhlaza luqukethe i-endotoxin nokunye ukungcola.Dlulisa i-I-DNA-equkethe isigaba samanzi eshubhu elisha futhilahla ungqimba olunamafutha.

Δ Izinga lokushisa phakathi ne-centrifugation kufanele libe ngaphezu kuka-25℃ njengoba ukuhlukaniswa kwesigaba okusebenzayo kungasebenzikwenzeka uma izinga lokushisa liphansi kakhulu.

Δ Uma ukuhlukaniswa kwesigaba kungasebenzi, izinga lokushisa le-centrifugation lingalungiswa libe ngu-30 ℃ futhiisikhathi se-centrifugation singanyuswa sibe yimizuzu eyi-15.

Δ Ungamunyi ungqimba olunamafutha aluhlaza okwesibhakabhaka njengoba luqukethe i-endotoxin nokunye ukungcola.

Indlela

Ukumiswa kabusha kwe-Lysis Neutralization

◇ Engeza u-7.5 ml Buffer P1

◇ Engeza u-7.5 ml Buffer P2

◇ Engeza u-7.5 ml Buffer P4

Ukususwa kwe-endotoxin

◇Engeza izikhathi ezingu-0. izikhathi ezingu-1 kunomthamo omkhulu we-Endotoxin Scavenger

Ukubopha nokuwasha

◇ Engeza izikhathi ezingu-0.5 zevolumu ye-isopropanol

◇ Engeza u-10 ml weBhafa PW