EndoFree Plasmid Maxi Kit
Setsi sena se loketse ho ntšoa ho tloha ho 150 - 300 ml ea tharollo ea baktheria e hlahisitsoeng bosiu bo le bong, ho sebelisoa mokhoa o ntlafalitsoeng oa SDS-alkaline lysis ho lyse baktheria.Sehlahisoa se sa hloekang se kopantsoe ka mokhoa o ikhethileng le Endotoxin Scavenger e ikhethang 'me e arotsoe ke centrifugation ho tlosa endotoxins.Joale, lera la silika la gel le khetha ho tlama ho plasmid DNA ka tharollo tlas'a maemo a letsoai le phahameng le pH e tlase.Sena se lateloa ke ho eketsoa ha sesepa sa ho hlatsoa ho tlosa litšila le likarolo tse ling tsa baktheria.Qetellong, sekontiri se nang le letsoai le tlaase, se nang le pH e phahameng se sebelisoa ho hlahisa DNA e hloekileng ea plasmid ho tloha lera la matrix la silicon.Lera la silika la gel le sebelisa lesela le khethehileng la adsorption, 'me phapang ea palo ea adsorption lipakeng tsa kholomo le kholomo e nyane haholo mme ho pheta-pheta ho lokile.Phenol, chloroform le li-reagents tse ling tse chefo ha li hlokehe, le mehato ea ho na ha ethanol.Sesebelisoa sena se ka sebelisoa ho ntša ka potlako 0.2 -1.5 mg ea DNA ea plasmid e hloekileng e phahameng, ka tekanyo ea ho ntša 80% -90%.Thepa e sebelisa mokhoa o ikhethileng oa ts'ebetso e tlosa endotoxin, likahare tsa endotoxin li tlase haholo mme phello ea phetisetso ea sele e ntle haholo.Plasma e ntšitsoeng e ka sebelisoa ka kotloloho ho tšilo ea enzyme, PCR, in vitro transcript, phetoho, tatellano le liteko tse ling tsa baeloji ea limolek'hule.
Maemo a polokelo
RNaseA e lokela ho bolokoa ho -30 ~ -15 ℃ 'me e tsamaise ka ≤0℃.
Endotoxin Scavenger e ka bolokoa ho 2 ~ 8 ℃ khoeli e le 'ngoe, e bolokiloe ho -30 ~ -15 ℃ bakeng sa polokelo ea nako e telele.'me e tsamaisoa ka ≤0℃ .
Likarolo tse ling li lokela ho bolokoa mocheso oa kamore (15 ~ 25 ℃) ebe li tsamaisoa ka mocheso oa kamore.
Likaroloana
| Likaroloana | 10RXNS |
| RNase A | 750 μL |
| Buffer P1 | 75 ml |
| Buffer P2 | 75 ml |
| Buffer P4 | 75 ml |
| Endotoxin Scavenger | 25 ml |
| Sekhahla sa PW | 2 × 22 di ml |
| Thibela TB | 20 ml |
| FastPure DNA Maxi Columns ( E 'ngoe le e 'ngoe ka har'a Tube ea Pokello ea 50ml) | 10 |
| Tube ea pokello e se nang Endotoxin | 2 × 5 |
RNaseA:10 mg/ml, e sebelisetsoang ho tlosa RNA.
Buffer P1:bacterial suspension buffer, eketsa RNaseA ho Buffer P1 pele o sebelisoa pele.
Buffer P2:baktheria lysis buffer (e nang le SDS/NaOH).
Buffer P4:sekoahelo se fokolang.
Endotoxin Scavenger:tlosa endotoxin ka katleho ho qotsulo e tala ea plasmid.
Buffer PW:hlatsoa buffer, eketsa molumo o boletsoeng oa ethanol pele u e sebelisa.
Buffer TB:elution buffer.
FastPure DNA Maxi Columns:plasmid DNA adsorption columns.
Li-tubes tsa pokello 50 ml:li-tubes tsa pokello ea filtrate.
Tube ea Pokello ea Endotoxin-Free:li-tubes tsa pokello ea DNA ea plasmid.
Lintho Tse Lokisitsoeng
Ethanol e felletseng, isopropanol, li-tubes tsa centrifuge tse 50 ml tse chitja tse tlase le 50 ml endotoxin- mahala.li-tubes tsa centrifuge.
Lisebelisoa
Sehlahisoa sena se loketse ho ntšoa ka bongata ba li-plasmids ho tloha ho 150 - 300 ml ea tharollo ea baktheria.setso ka bosiu bo le bong.
Mokhoa oa ho Leka
1. Nka 150 - 200 ml (e seng ho feta 300 ml) ea tharollo ea baktheria e hlahisitsoeng bosiu bo le bong le centrifugehoo e ka bang 11,000 rpm (12,000 × g) bakeng sa 1 - 2 min.Lahla matla a phahametseng tlhaho 'me u bokelle libaktheria.
Δ Ha o bokella ho feta 50 ml ea tharollo ea baktheria, libaktheria li ka bokelloa ka ho eketsa tharollo ea baktheria, centrifugation, ho lahla matla a holimo le mehato e meng ka har'a tube e tšoanang ea 50 ml.
makhetlo a mangata.
2. Kenya 7.5 ml ea Buffer P1 (ka kopo sheba hore na RNaseA e kentsoe ho Buffer P1) ho centrifuge.tube e nang le libaktheria ebe e kopanya hantle ka vortex kapa pipetting.
Δ Ho tsosolosoa ka ho feletseng ha libaktheria mohatong ona ho bohlokoa ho hlahisa, 'me ha hoa lokela ho ba le lihlopha tsa baktheria ka mor'a ho tsosolosoa.Haeba ho na le li-clumps tsa baktheria tse sa tsoakoang ka ho feletseng, li tla ama lysis, e leng se hlahisang chai e tlaase le bohloeki.Haeba OD600 ea tharollo ea baktheria ke 0.65, ho kgothaletswa hore 7.5 ml ya Buffer P1 e sebelisoe ha ho ntšoa ho 150 ml ea tharollo ea baktheria;ha OD600 e le 0.75, 8 ml ea Buffer P1 e lokela ho sebelisoa le meqolo ea Buffers P2 le P4 e lokela ho fetoloa ho latela.Haeba molumo oa tharollo ea baktheria o eketsoa ho 200 ml, ho kgothaletswa hore o be lebophahamo ba Buffers P1, P2, le P4 e eketsoe ka ho lekana.
3. Kenya 7.5 ml ea Buffer P2 ho ho emisoa ha baktheria ho tloha mohato oa 2 'me u kopanye ka bonolo ho ea holimo le tlase bakeng sa 6 - 8.linako le ho kenya mocheso oa kamore bakeng sa 4 - 5 min.
Δ Fetola ka bonolo ho kopanya hantle.Vortexing e tla baka karohano ea genomic DNA, e hlahisang likaroloana tsa DNA tsa genomic ho plasmid e ntšitsoeng.Ka nako ena, tharollo e fetoha viscous le translucent, e bontšang hore libaktheria li lysed ka ho feletseng.Nako ha ea lokela ho feta metsotso e 5 ho qoba tšenyo ea li-plasmids.Haeba tharollo e sa hlaka, ho ka ba le libaktheria tse ngata haholo tse bakanglysis e sa fellang, kahoo palo ea libaktheria e lokela ho fokotsoa ka nepo.
4. Eketsa 7.5 ml ea Buffer P4 ho ho emisoa ha baktheria ho tloha mohato oa 3 'me hang-hang u fetole ka bonolo 6 - 8 linako tse ling ho lumella tharollo ho fokotsa ka ho feletseng Buffer P2.Ka nako ena, flocculent precipitate e tšoeu e lokela ho hlaha.Centrifuge ka ho feta 11,000 rpm (12,000 × g) bakeng sa 10 - 15 min, pippette supernatant ka hloko e ncha ea 50 ml round-bottom centrifuge tube (e itokiselitseng), 'me u qobe.ho lakaletsa metsi a masoeu a phaphametseng.
Δ Eketsa Buffer P4 'me hang-hang u fetole ho kopanya hantle.Tlohela tube hore e eme ho fihlela pula e tšoeu e ajoa ka ho lekana ho pholletsa le tharollo ho thibela tlhahiso ea pula ea lehae e ka amang neutralization.Haeba ho se na junifomo tšoeu flocculent precipitate pele centrifugation le supernatant e sa hlaka ka mor'a centrifugation, tube e ka ba.centrifuged bakeng sa metsotso e meng e 5.
5. Eketsa 0. makhetlo a 1 ho feta (10% ea molumo o moholo, hoo e ka bang 2.2 ml) ea Endotoxin Scavenger ho ea ho supernatant ho tloha mohato oa 4 le ho khelosa ho kopanya.Beha tharollo ka bateng ea leqhoa kapa u kenye ka leqhoa le pshatlehileng (kapa sehatsetsing sa sehatsetsi) bakeng sa metsotso e 5 ho fihlela tharollo e fetoha ho tloha ho turbid ho ea ho hlaka le ho hlaka (kapa ho ntse ho le joalo.hanyenyane), 'me ka linako tse ling kopanya makhetlo a mangata.
Δ Kamora hore Endotoxin Scavenger e eketsoe ho matla a phahametseng tlhaho, matla a phahametseng a tla fetoha moferefere empasupernatant e lokela ho hlaka (kapa e be le litšila hanyenyane) ka mor'a ho pholile ka bateng ea leqhoa.
6. Ka mor'a hore supernatant e behoe mocheso oa kamore (> 25 ℃) bakeng sa 10 - 15 min, e tla fetoha lerata.mocheso oa eona o nyolohela mocheso oa kamore.Ebe supernatant e lokela ho khelosoa ho kopanya.
Δ Haeba mocheso oa kamore o le tlase kapa o batla ho fokotsa nako ea ho ntša, supernatant e ka kenngoa ka bateng ea metsi ea 37 ~ 42 ℃ bakeng sa 5 - 10 min mme mohato o latelang o ka etsoa ka mor'a supernatant.e ba morusu.
7. Centrifuge the supernatant hoo e ka bang 11,000 rpm (12,000 × g) bakeng sa metsotso e 10 mocheso oa kamore (mocheso o tlameha ho ba> 25 ℃) ho arola mohato.Karolo e kaholimo ea metsi e na le DNA ha karolo e tlase ea oli e boputsoa e na le endotoxin le litšila tse ling.FetisetsaDNA-e nang le mokhahlelo oa metsi ho ea ho tube e ncha lelahla lera le mafura.
Δ Thempereichara nakong ea centrifugation e tlameha ho ba ka holimo ho 25℃ kaha karohano e sebetsang e sa sebetseetsahala haeba mocheso o le tlase haholo.
Δ Haeba karohano ea karolo e sa sebetse, mocheso oa centrifugation o ka fetoloa ho 30 ℃ lenako ea centrifugation e ka eketsoa ho 15 min.
Δ Se ke oa monya lesela le nang le oli e putsoa kaha le na le endotoxin le litšila tse ling.
Mokhoa
Resuspension Lysis Neutralization
◇ Kenya 7.5 ml Buffer P1
◇ Kenya 7.5 ml Buffer P2
◇ Kenya 7.5 ml Buffer P4
Ho tlosoa ha endotoxin
◇Eketsa 0. makhetlo a 1 ho feta molumo o moholo oa Endotoxin Scavenger
Ho tlama le ho Hlatsoa
◇ Eketsa ka makhetlo a 0,5 bophahamo ba isopropanol
◇ Kenya 10 ml Buffer PW
◇ Kenya 10 ml Buffer PW
Elution
◇ Kenya 1 – 2 ml Buffer TB kapa Endotoxin-free ddH2O
