EndoFree Plasmid Maxi Kit
Iyi kit yakakodzera kutorwa kubva ku150 - 300 ml yebhakitiriya mhinduro yakasimudzwa usiku humwe, uchishandisa yakagadziridzwa SDS-alkaline lysis nzira ye lyse mabhakitiriya.Iyo isina kuchena inobvisa inosanganiswa yakasarudzika neyakasarudzika Endotoxin Scavenger uye inoparadzaniswa necentrifugation kubvisa endotoxins.Zvadaro, iyo silica gel membrane inosarudza inosunga kuplasmid DNA mune mhinduro pasi pemamiriro e-high-munyu uye pasi-pH.Izvi zvinoteverwa nekuwedzera kwewash buffer kubvisa tsvina nezvimwe zvinoumba utachiona.Pakupedzisira, yakaderera-munyu, yakakwirira-pH elution buffer inoshandiswa kuburitsa yakachena plasmid DNA kubva kune silicon matrix membrane.Iyo silica gel membrane inoshandisa yakakosha adsorption membrane, uye iyo adsorption chiyero musiyano pakati pekoramu nekoramu idiki kwazvo uye kudzokorora kwakanaka.Phenol, chloroform uye zvimwe zvine chepfu reagents hazvidiwe, uye zvakare haaite ethanol kunaya matanho.Iyi kit inogona kushandiswa kukurumidza kubvisa 0.2 -1.5 mg yeyakachena yepamusoro-copy plasmid DNA, ine chiyero chekubvisa che80% -90%.Iyo kit inoshandisa yakasarudzika maitiro formula inobvisa endotoxin, zvirimo endotoxin zvakaderera zvakanyanya uye sero transfection mhedzisiro yakanaka.Iyo plasmid yakabviswa inogona kushandiswa zvakananga mukuita enzyme digestion, PCR, in vitro transcription, shanduko, kutevedzana uye kumwe kuedza kwemamorekuru biology.
Kuchengetedza mamiriro
RNaseA inofanira kuchengetwa pa -30 ~ -15 ℃ uye kutakurwa pa ≤0 ℃ .
Endotoxin Scavenger inogona kuchengetwa pa2 ~ 8 ℃ kwemwedzi mumwe, yakachengetwa pa -30 ~ -15 ℃ yekuchengetedza kwenguva refu.uye kutakurwa pa ≤0℃.
Zvimwe zvikamu zvinofanirwa kuchengetwa pakamuri tembiricha (15 ~ 25 ℃) uye kutakurwa pakamuri tembiricha.
Zvikamu
| Zvikamu | 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 |
| FastPure DNA Maxi Columns ( Imwe neimwe mu50ml Collection Tube) | 10 |
| Endotoxin-isina Kuunganidzwa Tube | 2 × 5 |
RNaseA:10 mg/ml, inoshandiswa kubvisa RNA.
Buffer P1:bhakitiriya kumiswa buffer, wedzera RNaseA kuBuffer P1 usati watanga kushandisa.
Buffer P2:bhakitiriya lysis buffer (ine SDS/NaOH).
Buffer P4:neutralizing buffer.
Endotoxin Scavenger:nyatso bvisa endotoxin kubva kune isina kuchena plasmid yakatorwa.
Buffer PW:geza buffer, wedzera huwandu hwakatemwa hwe ethanol usati watanga kushandisa.
Buffer TB:elution buffer.
FastPure DNA Maxi Columns:plasmid DNA adsorption columns.
Kuunganidza Machubhu 50 ml:filtrate collection tubes.
Endotoxin-isina Kuunganidza Tube:plasmid DNA yekuunganidza machubhu.
Zvinhu Zvakagadzirirwa
Absolute ethanol, isopropanol, 50 ml kutenderera-pasi centrifuge machubhu uye 50 ml endotoxin- isinacentrifuge tubes.
Applications
Ichi chigadzirwa chakakodzera kubviswa kukuru kweplasmids kubva pa150 - 300 ml yebhakitiriya mhinduro.tsika usiku hwose.
Experiment Process
1. Tora 150 - 200 ml (kwete kupfuura 300 ml) yemushonga webhakitiriya wakagadzirwa nehusiku humwe uye centrifuge pa.anenge 11,000 rpm (12,000 × g) ye1 - 2 min.Rasa mashura uye unganidza mabhakitiriya.
Δ Paunenge uchiunganidza zvinopfuura 50 ml yemhinduro yebhakitiriya, mabhakitiriya anogona kuunganidzwa nekuwedzera mhinduro yebhakitiriya, centrifugation, kurasa iyo supernatant uye mamwe matanho mune imwecheteyo 50 ml chubhu ye.
kakawanda.
2. Wedzera 7.5 ml yeBuffer P1 (ndopota tarisa kana RNaseA yawedzerwa kuBuffer P1) kune centrifugechubhu ine mabhakitiriya uye sanganisa zvakakwana nevortex kana pipetting.
Δ Kudzoreredzwa kwakazara kwebhakitiriya mune iyi nhanho yakakosha kubereka, uye hapafanirwe kuve nemabhakitiriya clumps mushure mekumiswa.Kana pane zviputi zvebhakitiriya zvisina kunyatsosanganiswa, zvinokanganisa lysis, zvichiita kuti zvive zvishoma uye kuchena.Kana iyo OD600 yemushonga webhakitiriya iri 0.65, zvinokurudzirwa kuti 7.5 ml yeBuffer P1 ishandiswe pakubvisa kubva ku150 ml yebhakitiriya mhinduro;apo OD600 iri 0.75, 8 ml yeBuffer P1 inofanira kushandiswa uye mavhoriyamu eBuffers P2 neP4 anofanira kuchinjwa saizvozvo.Kana iyo vhoriyamu yebhakitiriya mhinduro yakawedzera kusvika 200 ml, zvinokurudzirwa kuti iyovhoriyamu yeBuffers P1, P2, uye P4 iwedzere kuenzana.
3. Wedzera 7.5 ml yeBuffer P2 kune bhakitiriya kuturika kubva padanho rechipiri uye sanganisa zvinyoro-nyoro kumusoro nekudzika kwe6 - 8.nguva uye incubate pakamuri tembiricha kwema 4 - 5 min.
Δ Pindura zvinyoro nyoro kuti usanganise zvakakwana.Vortexing ichakonzera genomic DNA kupatsanurwa, zvichikonzera genomic DNA zvimedu mu plasmid yakabviswa.Panguva ino, mhinduro inova viscous uye translucent, ichiratidza kuti mabhakitiriya akave akazara lysed.Nguva yacho haifaniri kudarika 5 min kuti udzivise kuparadzwa kweplasmids.Kana mhinduro isina kujeka, panogona kunge paine mabhakitiriya akawandisa anokonzeralysis isina kukwana, saka huwandu hwebhakitiriya hunofanira kuderedzwa zvakakodzera.
4. Wedzera 7.5 ml yeBuffer P4 kune kumiswa kwebhakitiriya kubva padanho rechitatu uye pakarepo shandura zvinyoro 6 - 8 nguva kuti mhinduro yacho iite kuti Buffer P2 iite zvishoma.Panguva ino, white flocculent precipitate inofanira kuonekwa.Centrifuge inopfuura 11,000 rpm (12,000 × g) kwema 10 - 15 min, nyatsopinza iyo supernatant mune itsva 50 ml round-bottom centrifuge chubhu (yakagadzirira wega), uye dzivirira.aspirate the white floating precipitate.
Δ Wedzera Buffer P4 uye pakarepo shandura kuti usanganise zvakanaka.Siya chubhu kuti imire kusvikira mvura yakachena yagovaniswa zvakaenzana mukati memhinduro kudzivirira kugadzirwa kwemvura yemunzvimbo iyo inogona kukanganisa neutralization.Kana pasina yunifomu chena flocculent precipitate pamberi pe centrifugation uye supernatant haina kujeka mushure me centrifugation, chubhu inogonacentrifuged kune imwe 5 min.
5. Wedzera 0. 1 nguva yevhoriyamu (10% yehuwandu hwepamusoro, inenge 2.2 ml) yeEndotoxin Scavenger kune supernatant kubva padanho rechina uye invert to mix.Isa mhinduro mubhavhu rechando kana kuisa muchando chakapwanyika (kana mufiriji mufiriji) kwemaminetsi mashanu kusvika mhinduro yachinja kubva kumarara kuenda kuchena uye pachena (kana kuchiri.zvishoma turbid), uye dzimwe nguva sanganisa kakawanda.
Δ Mushure mekunge Endotoxin Scavenger yawedzerwa kune iyo inoshamisa, iyo supernatant inozoita mhirizhonga asi iyosupernatant inofanira kujeka (kana zvishoma turbid) mushure mekutonhora mubhati rechando.
6. Mushure mekunge iyo supernatant yaiswa pakamuri tembiricha (>25 ℃) kwe10 - 15 min, inozoita mhirizhonga setembiricha yaro inowedzera kusvika mukamuri tembiricha.Ipapo iyo mashura inofanira kutenderedzwa kuti isanganise.
Δ Kana tembiricha yekamuri yakadzikira kana iwe uchida kudzikisa nguva yekubvisa, iyo supernatant inogona kuiswa mu 37 ~ 42 ℃ yekugezesa mvura ye5 - 10 min uye nhanho inotevera inogona kuitwa mushure meiyo supernatant.inoita mhirizhonga.
7. Centrifuge the supernatant pane 11,000 rpm (12,000 × g) kwemaminetsi gumi patembiricha yekamuri (tembiricha inofanira kuva>25℃) kuparadzanisa chikamu.Iyo yekumusoro aqueous chikamu ine DNA nepo yezasi yebhuruu ine mafuta chikamu layer ine endotoxin uye kumwe kusvibiswa.Transfer theDNA-ine aqueous chikamu kune itsva chubhu uyebvisa chidimbu chemafuta.
Δ The tembiricha panguva centrifugation inofanira kuva pamusoro 25 ℃ sezvo inoshanda chikamu kupatsanurwa kusingaitikuitika kana tembiricha yakaderera.
Δ Kana kupatsanurwa kwechikamu kusingashande, tembiricha ye centrifugation inogona kugadziridzwa kusvika 30 ℃ uye.nguva ye centrifugation inogona kuwedzerwa kusvika 15 min.
Δ Usayamwe bhuruu ine mafuta ebhuruu sezvo iine endotoxin uye kumwe kusvibiswa.
Mechanism
Resuspension Lysis Neutralization
◇ Wedzera 7.5 ml Buffer P1
◇ Wedzera 7.5 ml Buffer P2
◇ Wedzera 7.5 ml Buffer P4
Endotoxin kubvisa
◇Wedzera 0. ka1 pahuwandu hwepamusoro hweEndotoxin Scavenger
Kusunga uye Kuwacha
◇ Wedzera ka0,5 kuwanda kwehuwandu hweisopropanol
◇ Wedzera 10 ml Buffer PW
◇ Wedzera 10 ml Buffer PW
Elution
◇ Wedzera 1 - 2 ml Buffer TB kana Endotoxin-isina ddH2O
