EndoFree Plasmid Maxi Kit
Chidachi ndi choyenera kuchotsedwa pa 150 - 300 ml ya njira ya bakiteriya yomwe imabzalidwa usiku wonse, pogwiritsa ntchito njira yowonjezereka ya SDS-alkaline lysis kuti awononge mabakiteriya.Zosakaniza zopanda pake zimaphatikizidwa mosankhidwa ndi Endotoxin Scavenger yapadera ndikusiyanitsidwa ndi centrifugation kuchotsa endotoxins.Kenako, nembanemba ya gelisi ya silika imamangirira ku plasmid DNA mu njira yothetsera mchere wambiri komanso pH yochepa.Izi zimatsatiridwa ndi kuwonjezera zotchingira zochapira kuchotsa zonyansa ndi mabakiteriya ena.Pomaliza, chotchinga chamchere chochepa, chokhala ndi pH yayikulu chimagwiritsidwa ntchito kutulutsa DNA yoyera ya plasmid kuchokera ku nembanemba ya silicon matrix.Nembanemba ya silika ya gel imagwiritsa ntchito nembanemba yapadera yotsatsira, ndipo kusiyana kwa kuchuluka kwa adsorption pakati pa ndime ndi gawoli ndikochepa kwambiri ndipo kubwereza ndikwabwino.Phenol, chloroform ndi zinthu zina zapoizoni sizifunikanso, komanso masitepe a mpweya wa ethanol.zida izi angagwiritsidwe ntchito mofulumira wachotsa 0.2 -1.5 mg wa koyera mkulu-kope plasmid DNA, ndi mlingo m'zigawo za 80% -90%.Chidacho chimagwiritsa ntchito njira yapadera yochotsera endotoxin, zomwe zili mu endotoxin ndizochepa kwambiri ndipo mphamvu yopatsirana ma cell ndiyabwino kwambiri.Ma plasmid ochotsedwa atha kugwiritsidwa ntchito mwachindunji pakugaya kwa enzyme, PCR, in vitro transcript, kusintha, kutsatizana ndi kuyesa kwina kwa biology.
Zosungirako
RNaseA iyenera kusungidwa pa -30 ~ -15 ℃ ndikunyamulidwa pa ≤0 ℃.
Endotoxin Scavenger ikhoza kusungidwa pa 2 ~ 8 ℃ kwa mwezi umodzi, kusungidwa pa -30 ~ -15 ℃ posungira nthawi yayitalindi kunyamulidwa pa ≤0 ℃ .
Zigawo zina ziyenera kusungidwa kutentha (15 ~ 25 ℃) ndikunyamulidwa kutentha.
Zigawo
| Zigawo | Mtengo wa 10RXNS |
| Rnase A | 750 μl |
| Mtengo P1 | 75 ml pa |
| Mtengo P2 | 75 ml pa |
| Mtengo P4 | 75 ml pa |
| Endotoxin Scavenger | 25 ml pa |
| Mtengo PW | 2 × 22 ml |
| Chifuwa cha TB | 20 ml |
| FastPure DNA Maxi Columns (Iliyonse mu 50ml Collection Tube) | 10 |
| Endotoxin-Free Collection Tube | 2 × 5 pa |
RNaseA:10 mg/ml, yomwe imagwiritsidwa ntchito kuchotsa RNA.
Mtundu P1:bakiteriya kuyimitsidwa chitetezo, onjezani RNaseA ku Buffer P1 musanagwiritse ntchito koyamba.
Mtundu wa P2:bacterial lysis buffer (yokhala ndi SDS/NaOH).
Mtundu wa P4:neutralizing buffer.
Endotoxin Scavenger:Chotsani bwino endotoxin kuchokera ku plasmid yopanda pake.
Mtengo wa PW:Tsukani bafa, onjezani kuchuluka kwa ethanol musanayambe kugwiritsa ntchito koyamba.
Chifuwa cha TB:chotchinga chotchinga.
FastPure DNA Maxi Columns:plasmid DNA adsorption columns.
Machubu osonkhanitsira 50 ml:kusefera zosonkhanitsira machubu.
Tube Yosonkhanitsira Yopanda Endotoxin:machubu otolera a plasmid DNA.
Zida Zokonzekera
Mowa wamtheradi, isopropanol, 50 ml machubu ozungulira-pansi a centrifuge ndi 50 ml endotoxin wopandamachubu a centrifuge.
Mapulogalamu
Izi ndizoyenera kutulutsa kwakukulu kwa plasmids kuchokera ku 150 - 300 ml ya yankho la bakiteriya.chikhalidwe usiku.
Njira Yoyesera
1. Tengani 150 - 200 ml (osapitirira 300 ml) ya bakiteriya solution yolimidwa usiku wonse ndi centrifuge pa.pafupifupi 11,000 rpm (12,000 × g) kwa 1 - 2 min.Tayani zamatsenga ndikusonkhanitsa mabakiteriya.
Δ Potolera kupitilira 50 ml ya njira ya bakiteriya, mabakiteriya amatha kusonkhanitsidwa powonjezera njira ya bakiteriya, centrifugation, kutaya mphamvu yamphamvu ndi masitepe ena mu chubu lomwelo la 50 ml.
kangapo.
2. Onjezani 7.5 ml ya Buffer P1 (chonde onani ngati RNaseA yawonjezedwa ku Buffer P1) ku centrifugechubu chokhala ndi mabakiteriya ndikusakaniza bwino ndi vortex kapena pipetting.
Δ Kuyimitsidwa kwathunthu kwa mabakiteriya mu sitepe iyi ndikofunika kuti pakhale zokolola, ndipo sipayenera kukhala mabakiteriya oyambitsa pambuyo poyimitsidwa.Ngati pali mabakiteriya omwe sali osakanikirana bwino, zidzakhudza lysis, zomwe zimapangitsa kuti pakhale zokolola zochepa komanso zoyera.Ngati OD600 ya bakiteriya solution ndi 0,65, ndi bwino kuti 7.5 ml ya Buffer P1 igwiritsidwe ntchito pochotsa mu 150 ml ya njira ya bakiteriya;pamene OD600 ndi 0.75, 8 ml ya Buffer P1 iyenera kugwiritsidwa ntchito ndipo mavoliyumu a Buffers P2 ndi P4 ayenera kusinthidwa moyenerera.Ngati yankho la bakiteriya likuwonjezeka kufika 200 ml, ndi bwino kutikuchuluka kwa Buffers P1, P2, ndi P4 kuchulukitsidwa molingana.
3. Onjezani 7.5 ml ya Buffer P2 ku kuyimitsidwa kwa bakiteriya kuchokera ku sitepe 2 ndikusakaniza mofatsa mmwamba ndi pansi kwa 6 - 8.nthawi ndi kuulira kutentha kwa 4 - 5 min.
Δ Sinthani mofatsa kuti musakanize bwino.Vortexing imayambitsa kugawika kwa DNA, zomwe zimapangitsa kuti tizidutswa ta genomic DNA mu plasmid yotulutsidwa.Panthawiyi, yankho limakhala lowoneka bwino komanso lowoneka bwino, kusonyeza kuti mabakiteriya atsekedwa kwathunthu.Kutalika sikuyenera kupitirira 5 min kuti mupewe kuwonongeka kwa plasmids.Ngati yankho silikumveka bwino, pakhoza kukhala mabakiteriya ochulukirapo omwe angayambitselysis yosakwanira, kotero kuchuluka kwa mabakiteriya kuyenera kuchepetsedwa moyenera.
4. Onjezani 7.5 ml ya Buffer P4 ku kuyimitsidwa kwa bakiteriya kuchokera pa sitepe 3 ndipo nthawi yomweyo mutembenuzire mofatsa ka 6 - 8 kuti yankho lithetseretu Buffer P2.Panthawi imeneyi, nsonga zoyera za flocculent ziyenera kuwoneka.Centrifuge pafupifupi 11,000 rpm (12,000 × g) kwa 10 - 15 min, mosamala pippette ya supernatant mu chubu chatsopano cha 50 ml yozungulira-pansi (yokonzekera yokha), ndipo pewanifuna mvula yoyandama yoyera.
Δ Onjezani Buffer P4 ndipo nthawi yomweyo mutembenuzire kusakaniza bwino.Siyani chubu kuti chiyime mpaka mpweya woyera ugawidwe mofanana mu njira yothetsera mvula yam'deralo yomwe ingasokoneze kusokoneza.Ngati palibe yunifolomu woyera flocculent precipitate pamaso centrifugation ndi supernatant si bwino pambuyo centrifugation, chubu akhozacentrifuged kwa mphindi 5.
5. Onjezani 0. 1 nthawi ya voliyumu (10% ya voliyumu yamphamvu, pafupifupi 2.2 ml) ya Endotoxin Scavenger kupita ku supernatant kuchokera ku gawo 4 ndikutembenuza kuti musakanize.Ikani yankho mumadzi osambira oundana kapena ikani mu ayezi wophwanyidwa (kapena mufiriji mufiriji) kwa mphindi 5 mpaka yankho lisinthe kuchoka ku turbid kupita kumveka bwino (kapena kupitilirabe).turbid pang'ono), ndipo nthawi zina sakanizani kangapo.
Δ Endotoxin Scavenger ikawonjezedwa kwa wamphamvu, wamphamvuyo amakhala chipwirikiti komasupernatant iyenera kumveka bwino (kapena chipwirikiti pang'ono) ikazizira mumadzi oundana.
6. Mphamvu yamphamvu ikayikidwa pa firiji (>25 ℃) kwa 10 - 15 min, kumakhala chipwirikiti ngatikutentha kwake kumawonjezeka mpaka kutentha kwa chipinda.Kenako cham'mwambacho chiyenera kutembenuzidwa kuti chisakanize.
Δ Ngati kutentha kwa chipinda kuli kochepa kapena mukufuna kuchepetsa nthawi yochotsa, mphamvu yowonjezera ikhoza kukhazikitsidwa mumadzi osambira a 37 ~ 42 ℃ kwa 5 - 10 min ndipo sitepe yotsatira ikhoza kuchitidwa pambuyo pa supernatant.amakhala chipwirikiti.
7. Centrifuge the supernatant pafupifupi 11,000 rpm (12,000 × g) kwa mphindi 10 pa kutentha kwa firiji (kutentha kuyenera kukhala> 25 ℃) kupatutsa gawolo.Gawo lapamwamba lamadzi lili ndi DNA pomwe gawo lotsika lamafuta abuluu lili ndi endotoxin ndi zonyansa zina.Kusamutsa aDNA-munali amadzimadzi gawo ku chubu latsopano ndikutaya wosanjikiza mafuta.
Δ Kutentha pa centrifugation kuyenera kupitirira 25 ℃ monga kulekanitsa kogwira mtima sikumaterozimachitika ngati kutentha kuli kochepa kwambiri.
Δ Ngati kupatukana kwa gawo sikuli kothandiza, kutentha kwa centrifugation kumatha kusinthidwa kukhala 30 ℃ ndinthawi ya centrifugation akhoza ziwonjezeke kwa 15 min.
Δ Osamayamwa mafuta osanjikiza a buluu chifukwa ali ndi endotoxin ndi zonyansa zina.
Njira
Resuspension Lysis Neutralization
◇ Onjezani 7.5 ml Buffer P1
◇ Onjezani 7.5 ml Buffer P2
◇ Onjezani 7.5 ml Buffer P4
Kuchotsa endotoxin
◇Onjezani 0. kuchulukitsa ka 1 kuchuluka kwamphamvu kwa Endotoxin Scavenger
Kumanga ndi Kuchapa
◇ Onjezani kuwirikiza ka 0,5 kuchuluka kwa isopropanol
◇ Onjezani 10 ml Buffer PW
◇ Onjezani 10 ml Buffer PW
Elution
◇ Onjezani 1 – 2 ml Buffer TB kapena Endotoxin-free ddH2O
