EndoFree Plasmid Maxi Cov Khoom Siv
Cov khoom siv no tsim nyog rau kev rho tawm los ntawm 150 - 300 ml ntawm cov kab mob tshuaj tua kab mob thaum hmo ntuj, siv txoj kev txhim kho SDS-alkaline lysis kom lyse cov kab mob.Cov kua txiv hmab txiv ntoo yog xaiv ua ke nrog ib qho tshwj xeeb Endotoxin Scavenger thiab sib cais los ntawm centrifugation kom tshem tawm endotoxins.Tom qab ntawd, silica gel daim nyias nyias xaiv khi rau plasmid DNA hauv kev daws teeb meem nyob rau hauv cov xwm txheej ntawm cov ntsev thiab qis pH.Qhov no yog ua raws li kev sib ntxiv ntawm cov tshuaj ntxuav kom tshem tawm cov impurities thiab lwm yam kab mob.Thaum kawg, cov ntsev tsawg, siab pH elution tsis yog siv los qhia cov ntshiab plasmid DNA los ntawm silicon matrix membrane.Lub silica gel membrane siv tshwj xeeb adsorption membrane, thiab cov adsorption tus nqi sib txawv ntawm kem thiab kem yog heev me me thiab cov repeatability yog zoo.Phenol, chloroform thiab lwm yam tshuaj lom reagents tsis tas yuav tsum tau, thiab tsis yog ethanol nag lossis daus.Cov khoom siv no tuaj yeem siv los tshem tawm sai sai ntawm 0.2 -1.5 mg ntawm cov ntshiab high-copy plasmid DNA, nrog rau kev rho tawm ntawm 80% -90%.Cov khoom siv siv cov txheej txheem tshwj xeeb tshem tawm cov tshuaj endotoxin, cov ntsiab lus ntawm endotoxin tsis tshua muaj qis thiab cov txiaj ntsig ntawm cov cell transfection zoo heev.Cov plasmid rho tawm tuaj yeem siv ncaj qha rau hauv kev zom cov enzyme, PCR, hauv vitro transcription, hloov pauv, sequencing thiab lwm yam kev sim molecular biology.
Cia tej yam kev mob
RNaseA yuav tsum khaws cia ntawm -30 ~ -15 ℃ thiab thauj ntawm ≤0 ℃ .
Endotoxin Scavenger tuaj yeem khaws cia ntawm 2 ~ 8 ℃ rau ib hlis, khaws cia ntawm -30 ~ -15 ℃ rau kev cia ntevthiab thauj ntawm ≤0 ℃ .
Lwm cov khoom yuav tsum tau muab cia rau hauv chav tsev kub (15 ~ 25 ℃) thiab thauj hauv chav tsev kub.
Cheebtsam
| Cheebtsam | 10 RXNS |
| RNase A | 750l ua |
| Tsis P1 | 75ml ib |
| Tsis P2 | 75ml ib |
| Tsis P4 | 75ml ib |
| Endotoxin Scavenger | 25 ml ib |
| Buffer PW | 2 × 22 ml |
| Buffer TB | 20 ml ib |
| FastPure DNA Maxi Kab (Txhua hauv 50ml Collection Tube) | 10 |
| Endotoxin-dawb Collection Tube | 2 × 5 |
RNaseA:10 mg / ml, siv los tshem tawm RNA.
Buffer P1:Cov kab mob tshem tawm tsis zoo, ntxiv RNaseA rau Buffer P1 ua ntej siv.
Buffer P2:Cov kab mob lysis tsis tuaj yeem (muaj SDS / NaOH).
Tsis P4:neutralizing tsis.
Endotoxin Scavenger:zoo tshem tawm endotoxin los ntawm crude plasmid extract.
PW Buffer:ntxuav tsis, ntxiv qhov ntim ntawm ethanol ua ntej siv.
Buffer TB:elution tsis.
FastPure DNA Maxi Kab:plasmid DNA adsorption kab.
Collection Tubes 50 ml:filtrate collection tubes.
Endotoxin-dawb Collection Tube:plasmid DNA collection tubes.
Cov ntaub ntawv npaj
Tsis muaj ethanol, isopropanol, 50 ml puag ncig-hauv qab centrifuge raj thiab 50 ml endotoxin-dawbcentrifuge tubes.
Daim ntawv thov
Cov khoom no yog tsim rau loj-scale extraction ntawm plasmids los ntawm 150 - 300 ml ntawm cov kab mob tshuaj.kab lis kev cai hmo ntuj.
Kev sim txheej txheem
1. Noj 150 - 200 ml (tsis pub ntau tshaj 300 ml) ntawm cov kab mob tshuaj tua kab mob ib hmos thiab centrifuge ntawmtxog 11,000 rpm (12,000 × g) rau 1 - 2 min.Tshem tawm cov supernatant thiab sau cov kab mob.
Δ Thaum sau ntau tshaj 50 ml ntawm cov tshuaj tua kab mob, cov kab mob tuaj yeem sau tau los ntawm kev ntxiv cov tshuaj tua kab mob, centrifugation, pov tseg cov supernatant thiab lwm cov kauj ruam hauv tib 50 ml raj rau
ntau zaus.
2. Ntxiv 7.5 ml ntawm Buffer P1 (thov xyuas seb RNaseA puas tau ntxiv rau Buffer P1) rau lub centrifugeraj muaj cov kab mob thiab sib tov kom huv si los ntawm vortex los yog pipetting.
Δ Kev rov ua tiav ntawm cov kab mob hauv cov kauj ruam no yog qhov tseem ceeb rau kev tawm los, thiab yuav tsum tsis muaj kab mob tom qab rov ua dua.Yog tias muaj cov kab mob uas tsis sib xyaw ua ke, nws yuav cuam tshuam rau lysis, ua rau tsis muaj txiaj ntsig thiab purity.Yog hais tias OD600 ntawm cov kab mob tshuaj yog 0.65, nws raug pom zoo kom siv 7.5 ml ntawm Buffer P1 thaum rho tawm los ntawm 150 ml ntawm cov kab mob tshuaj;Thaum OD600 yog 0.75, 8 ml ntawm Buffer P1 yuav tsum tau siv thiab cov ntim ntawm Buffers P2 thiab P4 yuav tsum tau hloov raws li.Yog hais tias qhov ntim ntawm cov tshuaj tua kab mob yog nce mus rau 200 ml, nws raug pom zoo tias covQhov ntim ntawm Buffers P1, P2, thiab P4 tau nce proportionally.
3. Ntxiv 7.5 ml ntawm Buffer P2 mus rau cov kab mob tshem tawm los ntawm kauj ruam 2 thiab sib tov maj mam nce thiab nqis rau 6 - 8lub sij hawm thiab incubate nyob rau hauv chav tsev kub rau 4 - 5 min.
Δ Invert maj mam sib tov kom huv si.Vortexing yuav ua rau genomic DNA fragmentation, uas ua rau genomic DNA fragments nyob rau hauv lub plasmid rho tawm.Thaum lub sij hawm no, cov tshuaj ua viscous thiab translucent, qhia tias cov kab mob tau tag nrho lysed.Lub sijhawm yuav tsum tsis pub tshaj 5 feeb kom tsis txhob muaj kev puas tsuaj ntawm plasmids.Yog tias cov tshuaj tsis meej, tej zaum yuav muaj cov kab mob ntau dhau ua raulysis tsis tiav, yog li tus nqi ntawm cov kab mob yuav tsum raug txo kom tsim nyog.
4. Ntxiv 7.5 ml ntawm Buffer P4 rau cov kab mob tshem tawm los ntawm kauj ruam 3 thiab tam sim ntawd tig maj mam maj mam 6 - 8 zaug kom tso cai rau cov tshuaj kom tsis txhob muaj qhov tsis zoo ntawm Buffer P2.Lub sijhawm no, dawb flocculent precipitate yuav tsum tshwm sim.centrifuge ntawm ntau tshaj li ntawm 11,000 rpm (12,000 × g) rau 10 - 15 min, ua tib zoo pippette lub supernatant mus rau hauv ib tug tshiab 50 ml round-hauv qab centrifuge raj (tus kheej npaj), thiab zam.aspirate lub floating dawb precipitate.
Δ Ntxiv Buffer P4 thiab tam sim ntawd invert kom sib tov zoo.Tawm hauv lub raj kom sawv ntsug kom txog rau thaum cov nag lossis daus dawb tau muab faib ua tusyees thoob plaws hauv qhov kev daws teeb meem los tiv thaiv kev tsim cov nag lossis daus hauv zos uas tuaj yeem cuam tshuam rau qhov nruab nrab.Yog tias tsis muaj cov xim dawb flocculent precipitate ua ntej centrifugation thiab cov supernatant tsis meej tom qab centrifugation, lub raj tuaj yeem ua tau.centrifuged rau lwm 5 min.
5. Ntxiv 0. 1 zaug ntawm qhov ntim (10% ntawm lub ntim supernatant, txog 2.2 ml) ntawm Endotoxin Scavenger rau lub supernatant los ntawm kauj ruam 4 thiab invert mus rau tov.Muab cov tshuaj tso rau hauv ib da dej dej khov los yog ntxig rau hauv cov dej khov nab kuab (los yog lub tub yees lub tub yees) rau 5 feeb kom txog thaum cov tshuaj hloov los ntawm turbid mus rau ntshiab thiab pob tshab (los yog tseemme ntsis turbid), thiab qee zaus sib tov ob peb zaug.
Δ Tom qab cov Endotoxin Scavenger ntxiv rau lub supernatant, tus supernatant yuav dhau los ua turbid tab sissupernatant yuav tsum ua kom meej (los yog me ntsis turbid) tom qab txias hauv dej khov.
6. Tom qab lub supernatant muab tso rau ntawm chav tsev kub (> 25 ℃) rau 10 - 15 min, nws yuav turbid li.nws kub nce mus rau chav tsev kub.Tom qab ntawd tus supernatant yuav tsum tau inverted kom sib tov.
Δ Yog tias chav tsev kub qis dua lossis koj xav txo lub sijhawm rho tawm, lub supernatant tuaj yeem tsim nyob rau hauv 37 ~ 42 ℃ dej da dej rau 5 - 10 min thiab cov kauj ruam tom ntej tuaj yeem nqa tawm tom qab supernatant.ua turbid.
7. Centrifuge tus supernatant ntawm txog 11,000 rpm (12,000 × g) rau 10 min ntawm chav tsev kub (kub yuav tsum yog> 25 ℃) kom cais cov theem.Cov theem dej saum toj kawg nkaus muaj cov DNA thaum lub sij hawm qis qis oily theem txheej muaj endotoxin thiab lwm yam impurities.Hloov covDNA-muaj aqueous theem mus rau lub raj tshiab thiabpov tseg lub oily txheej.
Δ Qhov kub thaum lub sij hawm centrifugation yuav tsum tshaj 25 ℃ raws li theem kev sib cais tsis zootshwm sim yog tias qhov kub thiab txias qis dhau.
Δ Yog tias qhov kev sib cais theem tsis zoo, qhov kub ntawm centrifugation tuaj yeem hloov kho rau 30 ℃ thiabLub sijhawm ntawm centrifugation tuaj yeem nce mus rau 15 min.
Δ Tsis txhob nqus cov roj pleev xim xiav vim nws muaj endotoxin thiab lwm yam impurities.
Mechanism
Resuspension Lysis Neutralization
◇ Ntxiv 7.5 ml Buffer P1
◇ Ntxiv 7.5 ml Buffer P2
◇ Ntxiv 7.5 ml Buffer P4
Kev tshem tawm Endotoxin
◇ Ntxiv 0. 1 npaug ntawm qhov ntim supernatant ntawm Endotoxin Scavenger
Kev khi thiab ntxuav
◇ Ntxiv 0.5 npaug ntawm qhov ntim ntawm isopropanol
◇ Ntxiv 10 ml Buffer PW
◇ Ntxiv 10 ml Buffer PW
Elution
◇ Ntxiv 1 – 2 ml Buffer TB lossis Endotoxin-dawb ddH2O
