EndoFree Plasmid Maxi Kit
Ngwa a dabara adaba maka mmịpụta site na 150 - 300 ml nke ngwọta nje na-etolite n'otu abalị, na-eji usoro SDS-alkaline lysis ka mma iji kpochapụ nje bacteria.A na-ejikọta ihe mkpofu crude nke ọma na Endotoxin Scavenger pụrụ iche ma kewapụ ya site na centrifugation iji wepụ endotoxins.Mgbe ahụ, akpụkpọ ahụ silica gel na-ahọrọ na-ejikọta DNA plasmid na ngwọta n'okpuru ọnọdụ nke nnu dị elu na obere pH.Nke a na-esote mgbakwunye nke ihe nchekwa ihe iji wepụ adịghị ọcha na ihe ndị ọzọ na-akpata nje.N'ikpeazụ, a na-eji ihe ntanye ntanye dị ala, nke dị elu pH iji wepụ DNA plasmid dị ọcha site na akpụkpọ anụ silicon matrix.Akpụkpọ anụ silica gel na-eji akpụkpọ anụ adsorption pụrụ iche, na ọnụọgụ mgbasa ozi dị iche n'etiti kọlụm na kọlụm dị ntakịrị na nkwughachi dị mma.Achọghị Phenol, chloroform na ihe ndị ọzọ na-egbu egbu, ọ dịghịkwa usoro mmiri ozuzo ethanol.Enwere ike iji ngwa a wepụta ngwa ngwa 0.2 -1.5 mg nke DNA plasmid dị ọcha dị elu, yana ọnụego mmịpụta nke 80% -90%.Ngwa ahụ na-eji usoro usoro pụrụ iche na-ewepụ endotoxin, ọdịnaya nke endotoxin dị oke ala na mmetụta nnyefe cell dị mma.Enwere ike iji plasmid ewepụtara ozugbo na mgbaze enzyme, PCR, in vitro transcription, mgbanwe, usoro na nnwale ndị ọzọ dị ndụ.
Ọnọdụ nchekwa
Ekwesịrị ịchekwa RNaseA na -30 ~ -15 ℃ ma buru ya na ≤0℃.
Enwere ike ịchekwa Scavenger Endotoxin na 2 ~ 8 ℃ maka otu ọnwa, echekwara na -30 ~ -15 ℃ maka nchekwa ogologo oge.ma buru ya na ≤0℃.
Ekwesịrị ịchekwa ihe ndị ọzọ na ụlọ okpomọkụ (15 ~ 25 ℃) ma buru ya na ụlọ okpomọkụ.
Ngwa
| Ngwa | 10RXNS |
| RNase A | 750 μl |
| Ihe nchekwa P1 | 75 ml |
| Ihe nchekwa P2 | 75 ml |
| Ihe nchekwa P4 | 75 ml |
| Endotoxin Scavenger | 25 ml |
| Ihe nchekwa PW | 2 × 22 ml |
| Ihe nchekwa TB | 20 ml |
| FastPure DNA Maxi Columns (Onye ọ bụla na tube mkpokọta 50ml) | 10 |
| Tube mkpokọta enweghị Endotoxin | 2 × 5 |
RNaseA:10 mg/ml, eji wepụ RNA.
Ihe nchekwa P1:ihe nchekwa nkwusioru nje, tinye RNaseA na Buffer P1 tupu ojiji mbụ.
Ihe nchekwa P2:ihe nchekwa lysis nke nje (nwere SDS/NaOH).
Ihe nchekwa P4:ihe nchekwa neutralizing.
Endotoxin Scavenger:Wepụ endotoxin nke ọma na mpụta plasmid crude.
Ihe nchekwa PW:ịsacha ihe nchekwa, gbakwunye olu ethanol akọwapụtara tupu ojiji mbụ.
Ihe nchekwa TB:ihe nchekwa elution.
FastPure DNA Maxi Ogidi:ogidi adsorption DNA plasmid.
Tubes mkpokọta 50 ml:filtrate collection tubes.
Tube mkpokọta enweghị Endotoxin:plasmid DNA mkpokọta tubes.
Ngwa akwadoro
Ethanol zuru oke, isopropanol, 50 ml tubes centrifuge gbara gburugburu na 50 ml endotoxin-freecentrifuge tubes.
Ngwa
Ngwaahịa a dabara adaba maka nnukwu mmịpụta plasmids sitere na 150 - 300 ml nke ngwọta nje.omenala n'abali.
Usoro nnwale
1. Were 150 - 200 ml (ọ dịghị ihe karịrị 300 ml) nke ngwọta nje a kụrụ n'abali na centrifuge.ihe dịka 11,000 rpm (12,000 × g) maka 1 - 2 min.Tụfuo ihe dị elu ma kpokọta nje bacteria.
∆ Mgbe ị na-anakọta ihe karịrị 50 ml nke ngwọta nje, enwere ike ịnakọta nje ahụ site na ịgbakwunye ngwọta nje, centrifugation, tụfuo supernatant na usoro ndị ọzọ na otu 50 ml tube maka.
otutu oge.
2. Tinye 7.5 ml nke Buffer P1 (biko lelee ma etinyere RNaseA na Buffer P1) na centrifuge.tube nwere nje bacteria ma gwakọta nke ọma site na vortex ma ọ bụ pipetting.
Δ Mweghachi nke nje bacteria zuru oke na nzọụkwụ a dị oke mkpa iji nweta, na e kwesịghị inwe ihe mgbochi nje mgbe a maliteghachiri.Ọ bụrụ na e nwere nje clumps nke na-adịghị agwakọta nke ọma, ọ ga-emetụta lysis, na-akpata obere mkpụrụ na ịdị ọcha.Ọ bụrụ na OD600 nke ngwọta nje bụ 0.65, a na-atụ aro ka 7.5 ml nke Buffer P1 mee ihe mgbe ị na-ewepụta na 150 ml nke ngwọta nje;mgbe OD600 bụ 0.75, 8 ml nke Buffer P1 kwesịrị iji ya na mpịakọta nke Buffers P2 na P4 kwesịrị ịgbanwe ya.Ọ bụrụ na ọnụ ọgụgụ nke ngwọta nje na-abawanye ruo 200 ml, a na-atụ aro kaA ga-abawanye olu nke Buffers P1, P2, na P4 n'otu n'otu.
3. Tinye 7.5 ml nke Buffer P2 na nkwusioru nke nje bacteria site na nzọụkwụ 2 wee jikọta nwayọọ elu na ala maka 6 - 8ugboro na incubate na ụlọ okpomọkụ maka 4 - 5 min.
Δ Tụgharịa nwayọ ka ị gwakọta nke ọma.Vortexing ga-eme ka DNA nke mkpụrụ ndụ ihe nketa, na-ebute iberibe DNA genomic na plasmid ewepụtara.N'oge a, ihe ngwọta na-aghọ viscous na translucent, na-egosi na nje bacteria na-e zuru ezu lysed.Ogologo oge ekwesịghị gafere 5 min iji zere mbibi nke plasmids.Ọ bụrụ na ngwọta adịghị edozi, enwere ike ịnwe ọtụtụ nje bacteria na-ebute yalysis na-ezughị ezu, ya mere a ga-ebelata ọnụ ọgụgụ nke nje bacteria n'ụzọ kwesịrị ekwesị.
4. Tinye 7.5 ml nke Buffer P4 na nkwụsị nke nje bacteria site na nzọụkwụ 3 wee tụgharịa nwayọọ nwayọọ ugboro 6 - 8 iji mee ka ngwọta ahụ kwụsị kpamkpam Buffer P2.N'oge a, ihe na-acha ọcha flocculent kwesịrị ịpụta.Centrifuge n'ihe dị ka 11,000 rpm (12,000 × g) maka 10 - 15 min, jiri nlezianya pịgharịa ihe dị elu n'ime tube centrifuge ọhụrụ 50 ml gbara gburugburu (nke jikwa onwe ya), ma zere.aspirate na-ese n'elu mmiri na-acha ọcha.
Δ Tinye ihe nchekwa P4 wee tụgharịa ozugbo ka ọ gwakọta nke ọma.Hapụ tube ahụ ka ọ guzoro ruo mgbe a na-ekesa ihe na-acha ọcha ọcha n'otu oge na ngwọta niile iji gbochie mmepụta nke mmiri ozuzo mpaghara nke nwere ike imetụta nkwụsịtụ.Ọ bụrụ na ọ dịghị edo edo flocculent precipitate tupu centrifugation na supernatant adịghị anya mgbe centrifugation, tube nwere ike ịbụ.centrifud maka nkeji 5 ọzọ.
5. Tinye 0. 1 ugboro olu (10% nke olu dị elu, ihe dị ka 2.2 ml) nke Endotoxin Scavenger na onye na-emepụta ihe site na nzọụkwụ 4 ma tụgharịa ka mix.Debe ihe ngwọta n'ime mmiri ịsa ahụ ma ọ bụ tinye n'ime ice a gwepịara egwepịa (ma ọ bụ friji friji) maka 5 min ruo mgbe ngwọta gbanwere site na turbid ka ọ pụta ìhè na nke ọma (ma ọ bụ ka ọ dị.ubé turbid), na mgbe ụfọdụ mix ọtụtụ ugboro.
∆ Mgbe agbakwunyere Scavenger Endotoxin na onye na-achị achị, onye na-ahụ maka ya ga-aghọ turbid manasupernatant kwesịrị ịpụta ìhè (ma ọ bụ ntakịrị turbid) mgbe ọ dị jụụ na bat ice.
6. Mgbe supernatant etinye na ụlọ okpomọkụ (> 25 ℃) maka 10 - 15 min, ọ ga-aghọ turbid dị ka.okpomọkụ ya na-abawanye ruo ụlọ okpomọkụ.Mgbe ahụ, a ga-atụgharị isi ala ka ọ gwakọta.
∆ Ọ bụrụ na okpomọkụ ụlọ dị ala ma ọ bụ na ịchọrọ ibelata oge mmịpụta, enwere ike itinye supernatant n'ime mmiri ịsa mmiri 37 ~ 42 ℃ maka 5 - 10 min ma enwere ike ịme usoro ọzọ mgbe supernatant gasịrị.na-aghọ turbid.
7. Centrifuge supernatant na ihe dịka 11,000 rpm (12,000 × g) maka 10 min na ụlọ okpomọkụ (okpomọkụ ga-abụ> 25 ℃) iji kewaa oge.Akụkụ mmiri dị elu nwere DNA ebe oyi akwa mmanụ na-acha anụnụ anụnụ nwere endotoxin na adịghị ọcha ndị ọzọ.Nyefee nkeDNA-nwere aqueous agba na tube ọhụrụ natufuo akwa mmanu mmanu.
Δ The okpomọkụ n'oge centrifugation ga-n'elu 25 ℃ dị irè na-adọ nkewa adịghịeme ma ọ bụrụ na okpomọkụ dị oke ala.
Δ Ọ bụrụ na nkewa nke oge adịghị arụ ọrụ, enwere ike gbanwee okpomọkụ centrifugation na 30 ℃ naoge nke centrifugation nwere ike ịbawanye ruo 15 min.
∆Anụla akwa mmanụ na-acha anụnụ anụnụ n'ihi na o nwere endotoxin na adịghị ọcha ndị ọzọ.
Mechanism
Mweghachi nke Lysis Neutralization
◇ Tinye 7.5 ml ihe nchekwa P1
◇ Tinye 7.5 ml ihe nchekwa P2
◇ Tinye 7.5 ml ihe nchekwa P4
Mwepụ Endotoxin
◇ Tinye 0. 1 ugboro oke nke Endotoxin Scavenger
Ejikọ na ịsa ahụ
◇ Tinye 0,5 ugboro olu isopropanol
◇ Tinye 10 ml ihe nchekwa PW
◇ Tinye 10 ml ihe nchekwa PW
Elution
◇ Tinye 1 - 2 ml ihe mgbochi TB ma ọ bụ ddH2O na-enweghị Endotoxin
