EndoFree Plasmid Maxi Kit
Iki gikoresho gikwiranye no kuvana miriyoni 150 - 300 z'umuti wa bagiteri ziterwa nijoro, ukoresheje uburyo bwiza bwa SDS-alkaline lysis bwo gutera bagiteri.Ibicuruzwa bitavanze byahujwe hamwe na Endotoxin idasanzwe ya Scavenger hanyuma igatandukanywa na centrifugation kugirango ikureho endotoxine.Hanyuma, silika gel membrane ihitamo guhuza ADN ya plasmid mugisubizo mugihe cyumunyu mwinshi na pH nkeya.Ibi bikurikirwa no kongeramo buffer kugirango ukureho ubudahangarwa nibindi bice bya bagiteri.Ubwanyuma, umunyu muke, mwinshi-pH wohanagura bikoreshwa mugukuraho ADN yuzuye ya plasmide ivuye muri silicon matrix membrane.Silica gel membrane ikoresha adsorption idasanzwe, kandi ingano ya adsorption itandukanya inkingi ninkingi ni nto cyane kandi gusubiramo nibyiza.Fenol, chloroform nizindi reagent zifite ubumara ntibisabwa, kandi ntanintambwe yimvura ya Ethanol.Iki gikoresho kirashobora gukoreshwa mugukuramo vuba 0.2 -1.5 mg ya ADN yuzuye ya kopi ya plasmid ADN, hamwe nogukuramo 80% -90%.Igikoresho gikoresha formulaire idasanzwe ikuraho endotoxine, ibiri muri endotoxine ni bike cyane kandi ingaruka zo kwanduza selile ni nziza.Plasmid yakuweho irashobora gukoreshwa muburyo butaziguye mu igogorwa rya enzyme, PCR, mu kwandukura vitro, guhinduka, gukurikiranya hamwe nubundi bushakashatsi bwibinyabuzima bwa molekile.
Imiterere yo kubika
RNaseA igomba kubikwa kuri -30 ~ -15 ℃ ikajyanwa kuri ≤0 ℃.
Endotoxin Scavenger irashobora kubikwa kuri 2 ~ 8 ℃ ukwezi kumwe, ikabikwa kuri -30 ~ -15 ℃ kubikwa igihe kirekireno gutwarwa kuri ≤0 ℃.
Ibindi bice bigomba kubikwa mubushyuhe bwicyumba (15 ~ 25 ℃) kandi bigatwarwa mubushyuhe bwicyumba.
Ibigize
| Ibigize | 10RXNS |
| RNase A. | 750 μL |
| Buffer P1 | 75 ml |
| Buffer P2 | 75 ml |
| Buffer P4 | 75 ml |
| Endotoxin Scavenger | 25 ml |
| Buffer PW | 2 × 22 ml |
| Buffer TB | 20 ml |
| Inkingi yihuta ya ADN ya Maxi (Buri kimwe muri 50ml Ikusanyirizo) | 10 |
| Endotoxin-Yubusa | 2 × 5 |
RNaseA:10 mg / ml, ikoreshwa mu gukuraho RNA.
Buffer P1:guhagarika bagiteri, ongera RNaseA kuri Buffer P1 mbere yo gukoresha bwa mbere.
Buffer P2:bagiteri ya lysis buffer (irimo SDS / NaOH).
Buffer P4:kutabogama.
Endotoxin Scavenger:kuvanaho neza endotoxine mumashanyarazi ya plasmid.
Buffer PW:koza buffer, ongeramo ingano yagenwe ya Ethanol mbere yo gukoresha bwa mbere.
Buffer TB:buffer.
Inkingi yihuta ya ADN Maxi Inkingi:plasmid ADN ya adsorption inkingi.
Amashanyarazi yo gukusanya 50 ml:Akayunguruzo.
Endotoxin idafite icyegeranyo cya Tube:plasmid ADN yo gukusanya.
Ibikoresho Byateguwe
Ethanol yuzuye, isopropanol, ml 50 kuzenguruka-munsi ya centrifuge tubi na ml 50 endotoxine-centrifuge tubes.
Porogaramu
Iki gicuruzwa gikwiranye no gukuramo plasmide nini kuva kuri 150 - 300 ml yumuti wa bagiteriumuco nijoro.
Inzira y'Ubushakashatsi
1. Fata 150 - 200 ml (itarenze 300 ml) yumuti wa bagiteri utera ijoro hamwe na centrifuge kurihafi 11,000 rpm (12,000 × g) kuminota 1 - 2.Hagarika ndengakamere hanyuma ukusanye bagiteri.
Δ Iyo ukusanyije miriyoni zirenga 50 z'umuti wa bagiteri, bagiteri irashobora gukusanywa hiyongereyeho umuti wa bagiteri, centrifugation, ukajugunya ndengakamere nizindi ntambwe mumitwe imwe ya ml 50 kuri
inshuro nyinshi.
2. Ongeramo 7.5 ml ya Buffer P1 (nyamuneka reba niba RNaseA yongerewe muri Buffer P1) kuri centrifugeumuyoboro urimo bagiteri hanyuma ukavanga neza na vortex cyangwa pipetting.
Guhagarika burundu za bagiteri muri iyi ntambwe ni ingenzi cyane kubyara umusaruro, kandi ntihakagombye kubaho uduce twa bagiteri nyuma yo guhagarikwa.Niba hari udukoko twa bagiteri tutavanze neza, bizagira ingaruka kuri lysis, bivamo umusaruro muke nubuziranenge.Niba OD600 yumuti wa bagiteri ari 0,65, birasabwa ko miriyoni 7,5 za Buffer P1 zikoreshwa mugihe zivuye muri ml 150 yumuti wa bagiteri;mugihe OD600 ari 0,75, ml 8 ya Buffer P1 igomba gukoreshwa kandi ingano ya Buffers P2 na P4 igomba guhinduka.Niba ingano yumuti wa bagiteri yiyongereye kuri 200 ml, birasabwa koingano ya Buffers P1, P2, na P4 yongerewe uko bikwiye.
3. Ongeramo 7.5 ml ya Buffer P2 kuri bagiteri ihagarikwa kuva ku ntambwe ya 2 hanyuma uvange witonze hejuru no hepfo kuri 6 - 8inshuro no gushiramo ubushyuhe bwicyumba kuminota 4 - 5 min.
Hindura witonze kugirango uvange neza.Vortexing izatera ADN genomic gucamo ibice, bivamo ibice bya ADN bya ADN muri plasmide yakuwe.Muri iki gihe, igisubizo kiba cyiza kandi cyoroshye, byerekana ko bagiteri zatewe neza.Ikiringo ntigishobora kurenza min 5 kugirango wirinde gusenya plasmide.Niba igisubizo kidasobanutse, hashobora kubaho bagiteri nyinshi bikavamolysis ituzuye, bityo umubare wa bagiteri ugomba kugabanuka uko bikwiye.
4. Ongeramo 7.5 ml ya Buffer P4 muguhagarika kwa bagiteri kuva ku ntambwe ya 3 hanyuma uhite uhinduranya witonze inshuro 6 - 8 kugirango ubone igisubizo kibangamire rwose Buffer P2.Muri iki gihe, imvura yera ya flocculent igomba kugaragara.Centrifuge hafi ya 11,000 rpm (12,000 × g) muminota 10 - 15, witonze witondere supernatant mumashanyarazi mashya ya ml 50 azenguruka-munsi ya centrifuge (yiteguye), kandi wirindekwifuza imvura yera ireremba.
Δ Ongeramo Buffer P4 hanyuma uhite uhindura kugirango uvange neza.Kureka umuyoboro uhagarara kugeza igihe imvura yera igabanijwe neza mugisubizo kugirango wirinde umusaruro wimvura yaho ishobora kugira aho ibogamiye.Niba nta flokculent yera ihari mbere ya centrifugation kandi ndengakamere ntibisobanutse nyuma ya centrifugation, umuyoboro urashoboracentrifuged kuminota 5 min.
5. Ongeramo 0. inshuro 1 ingano (10% yubunini bwindengakamere, hafi 2,2 ml) ya Endotoxin Scavenger kuri supernatant kuva kuntambwe ya 4 hanyuma uhindure kuvanga.Shira igisubizo mubwogero bwa barafu cyangwa shyiramo urubura rwajanjaguwe (cyangwa firigo ya firigo) muminota 5 kugeza igisubizo gihindutse kiva mumivurungano kigaragara neza kandi kiboneye (cyangwa biracyaza)gato), kandi rimwe na rimwe kuvanga inshuro nyinshi.
Δ Nyuma ya Endotoxin Scavenger yongewe kuri ndengakamere, ndengakamere izahinduka akajagari ariko thendengakamere igomba gusobanuka (cyangwa ihungabana gato) nyuma yo gukonjesha mu bwogero bwa barafu.
6. Nyuma yuko ndengakamere ishyizwe mubushyuhe bwicyumba (> 25 ℃) muminota 10 - 15, bizahinduka akajagari nkukoubushyuhe bwayo bwiyongera kubushyuhe bwicyumba.Noneho super- superant igomba guhindurwa kugirango ivange.
Δ Niba ubushyuhe bwicyumba buri hasi cyangwa ushaka kugabanya igihe cyo kuvoma, ndengakamere irashobora gushirwa mubwogero bwamazi 37 ~ 42 min muminota 5 - 10 min hanyuma intambwe ikurikira irashobora gukorwa nyuma yindengakamere.ihinduka akajagari.
7. Centrifuge ndengakamere nka 11,000 rpm (12,000 × g) muminota 10 mubushyuhe bwicyumba (ubushyuhe bugomba kuba> 25 ℃) kugirango utandukanye icyiciro.Icyiciro cyo hejuru cyamazi kirimo ADN mugihe igice cyo hasi cyamavuta yubururu kirimo endotoxine nibindi byanduye.KwimuraADN irimo icyiciro cyamazi kumuyoboro mushya kandiguta urwego rwamavuta.
Δ Ubushyuhe mugihe cya centrifugation bugomba kuba hejuru ya 25 ℃ nkuko gutandukana kwicyiciro kutabikorabibaho niba ubushyuhe buri hasi cyane.
Δ Niba icyiciro cyo gutandukana kidakorwa neza, ubushyuhe bwa centrifugation burashobora guhinduka kuri 30 ℃ naigihe cya centrifugation gishobora kwiyongera kugeza 15 min.
Δ Ntunywe amavuta yubururu yubururu kuko arimo endotoxine nibindi byanduye.
Urwego
Guhagarika Lysis Kutabogama
◇ Ongeramo 7.5 ml Buffer P1
◇ Ongeramo 7.5 ml Buffer P2
◇ Ongeramo 7.5 ml Buffer P4
Gukuraho Endotoxine
Ongeraho 0. inshuro 1 ingano ndengakamere ya Endotoxin Scavenger
Guhambira no Gukaraba
◇ Ongeramo inshuro 0.5 ingano ya isopropanol
◇ Ongeramo ml 10 Buffer PW
◇ Ongeramo ml 10 Buffer PW
Gukuraho
◇ Ongeramo 1 - 2 ml Buffer TB cyangwa Endotoxine idafite ddH2O
