EndoFree Plasmid Maxi Kit
He kūpono kēia pahu no ka unuhi ʻana mai ka 150 - 300 ml o ka hopena bacteria i hoʻoulu ʻia i ka pō, me ka hoʻohana ʻana i kahi ala SDS-alkaline lysis i hoʻomaikaʻi ʻia e lyse i ka bacteria.Hoʻohui pū ʻia ka ʻōpala ʻōpala me kahi Endotoxin Scavenger a hoʻokaʻawale ʻia e ka centrifugation e wehe i nā endotoxins.A laila, paʻa ka silica gel membrane i ka plasmid DNA i ka hopena ma lalo o nā kūlana o ka paʻakai kiʻekiʻe a me ka pH haʻahaʻa.Hoʻopili ʻia kēia me ka hoʻohui ʻana i ka mea holoi holoi no ka wehe ʻana i nā haumia a me nā mea bacteria ʻē aʻe.ʻO ka hope, hoʻohana ʻia kahi paʻakai haʻahaʻa, pH kiʻekiʻe elution buffer e hoʻoheheʻe i ka DNA plasmid maʻemaʻe mai ka membrane matrix silicon.Hoʻohana ka membrane silica gel i ka membrane adsorption kūikawā, a ʻo ka nui o ka adsorption ʻokoʻa ma waena o ke kolamu a me ke kolamu he liʻiliʻi loa a maikaʻi ka hana hou.ʻAʻole koi ʻia ʻo Phenol, chloroform a me nā mea ʻawaʻawa ʻē aʻe, a ʻaʻole hoʻi nā ʻanuʻu ethanol precipitation.Hiki ke hoʻohana ʻia kēia kit e wehe koke i ka 0.2 -1.5 mg o ka DNA plasmid kope kiʻekiʻe maʻemaʻe, me ka helu unuhi o 80% -90%.Hoʻohana ka pahu i kahi ʻano kaʻina hana kūʻokoʻa e wehe i ka endotoxin, haʻahaʻa loa ka ʻike o ka endotoxin a maikaʻi loa ka hopena o ka hoʻololi cell.Hiki ke hoʻohana pololei ʻia ka plasmid i unuhi ʻia i ka ʻeli ʻana o ka enzyme, PCR, in vitro transcription, hoʻololi, sequencing a me nā hoʻokolohua biology molecular ʻē aʻe.
Nā kūlana mālama
Pono e mālama ʻia ʻo RNaseA ma -30 ~ -15 ℃ a lawe ʻia ma ≤0 ℃.
Hiki ke mālama ʻia ka Endotoxin Scavenger ma 2 ~ 8 ℃ no hoʻokahi mahina, mālama ʻia ma -30 ~ -15 ℃ no ka mālama lōʻihi.a lawe ʻia ma ≤0 ℃ .
Pono e mālama ʻia nā ʻāpana ʻē aʻe ma ke ana wela (15 ~ 25 ℃) a lawe ʻia i ka mahana wela.
Nā ʻāpana
| Nā ʻāpana | 10RXNS |
| ʻO RNase A | 750 μL |
| Palena P1 | 75 ml |
| Palena P2 | 75 ml |
| Palena P4 | 75 ml |
| ʻO ka Endotoxin Scavenger | 25 ml |
| Hoopaa PW | 2 × 22 ml |
| Hoʻopaʻa TB | 20 ml |
| ʻO nā kolamu ʻo FastPure DNA Maxi (ʻO kēlā me kēia i loko o kahi pahu hōʻiliʻili 50ml) | 10 |
| Paipu Ohi ʻAʻohe Endotoxin | 2 × 5 |
RNaseA:10 mg/ml, hoʻohana ʻia e wehe i ka RNA.
Hoʻopaʻa P1:paʻa hoʻokuʻu bacteria, hoʻohui i ka RNaseA i ka Buffer P1 ma mua o ka hoʻohana mua ʻana.
Hoʻopaʻa P2:pale lysis bacteria (loaʻa iā SDS/NaOH).
Hoʻopaʻa P4:pale pale pale.
Endotoxin Scavenger:e wehe pono i ka endotoxin mai ka plasmid extract.
Hoʻopaʻa PW:holoi holoi, hoʻohui i ka nui o ka ethanol ma mua o ka hoʻohana mua ʻana.
Hoʻopaʻa TB:elution buffer.
Nā kolamu Maxi DNA maʻemaʻe wikiwiki:plasmid DNA adsorption kolamu.
Nā paipu hōʻiliʻili 50 ml:nā paipu ʻohi kānana.
Paipu Ohi ʻAʻohe Endotoxin:nā paipu hōʻiliʻili DNA plasmid.
Nā mea i hoʻomākaukau ʻia
Ethanol piha, isopropanol, 50 ml mau paipu centrifuge a puni lalo a me 50 ml endotoxin-noa.nā paipu centrifuge.
Nā noi
He kūpono kēia huahana no ka unuhi nui ʻana o nā plasmid mai 150 - 300 ml o ka hopena bacterial.moʻomeheu i ka pō.
Kaʻina hoʻokolohua
1. E lawe i 150 - 200 ml (ʻaʻole ʻoi aku ma mua o 300 ml) o ka hopena bacteria i hoʻoulu ʻia i ka pō a me ka centrifuge mama kahi o 11,000 rpm (12,000 × g) no 1 – 2 min.E hoʻolei i ka supernatant a hōʻiliʻili i ka bacteria.
Δ Ke hōʻiliʻili ʻoi aku ma mua o 50 ml o ka hopena bacteri, hiki ke hōʻiliʻili ʻia ka bacteria ma ka hoʻohui ʻana i ka hopena bacterial, centrifugation, hoʻolei i ka supernatant a me nā ʻanuʻu ʻē aʻe i loko o ka pahu like 50 ml no
mau manawa.
2. E hoʻohui i 7.5 ml o Buffer P1 (e ʻoluʻolu e nānā inā ua hoʻohui ʻia ʻo RNaseA i ka Buffer P1) i ka centrifuge.paipu i loko o ka bacteria a hui pono me ka vortex a i ʻole pipetting.
Δ He mea koʻikoʻi ka hoʻihoʻi hou ʻana o ka hua bacteria ma kēia ʻanuʻu, a ʻaʻole pono e loaʻa nā puʻupuʻu bacteria ma hope o ka hoʻokuʻu ʻana.Inā loaʻa nā pūpū bacteria ʻaʻole i hui maikaʻi ʻia, e hoʻopilikia ia i ka lysis, e hopena i ka haʻahaʻa a me ka maʻemaʻe.Inā he 0.65 ka OD600 o ka hopena bacteria, pono e hoʻohana ʻia ka 7.5 ml o Buffer P1 i ka lawe ʻana mai 150 ml o ka hopena bacteria;inā he 0.75 ka OD600, pono e hoʻohana ʻia he 8 ml o ka Buffer P1 a e hoʻololi ʻia ka nui o nā Buffers P2 a me P4.Inā hoʻonui ʻia ka nui o ka hopena bacteria i 200 ml, ʻoi aku ka maikaʻiHoʻonui ʻia ka nui o nā Buffer P1, P2, a me P4 ma ke ʻano like.
3. E hoʻohui i 7.5 ml o ka Buffer P2 i ka hoʻokuʻu ʻia o ka maʻi bacteria mai ka ʻanuʻu 2 a hui mālie i luna a i lalo no 6 – 8.manawa a incubate ma ka lumi wela no 4 - 5 min.
Δ Huli malie e hui pono.ʻO ka Vortexing ke kumu i ka ʻāpana DNA genomic, ka hopena i nā ʻāpana DNA genomic i ka plasmid i unuhi ʻia.I kēia manawa, lilo ka hoʻonā ʻana i ka viscous a translucent, e hōʻike ana ua hoʻopiha piha ʻia ka bacteria.ʻAʻole pono ka lōʻihi ma mua o 5 min e pale i ka luku ʻana i nā plasmids.Inā ʻaʻole maopopo ka hoʻonā ʻana, nui paha ka hua bacteriaka lysis piha ʻole, no laila pono e hoʻemi ʻia ka nui o ka bacteria.
4. E hoʻohui i 7.5 ml o ka Buffer P4 i ka hoʻokuʻu ʻia ʻana o ka maʻi bacteria mai ka ʻanuʻu 3 a hoʻohuli mālie me ka mālie 6 - 8 manawa e ʻae i ka hopena e hoʻopau loa i ka Buffer P2.I kēia manawa, pono e ʻike ʻia nā ʻeke flocculent keʻokeʻo.ʻO ka Centrifuge ma kahi o 11,000 rpm (12,000 × g) no 10 - 15 min, e hoʻopili pono i ka supernatant i loko o kahi paipu centrifuge 50 ml a puni (hoʻomākaukau ponoʻī), a pale aku.aspirate the floating precipitate keʻokeʻo.
Δ Hoʻohui i ka Buffer P4 a hoʻohuli koke e hui maikaʻi.E waiho i ka paipu e kū a hiki i ka māhele like ʻana o ka ua keʻokeʻo i loko o ka hoʻonā e pale ai i ka hana ʻana o ka ua kūloko i hiki ke hoʻopilikia i ka neutralization.Inā ʻaʻohe ʻano like ʻole keʻokeʻo flocculent precipitate ma mua o ka centrifugation a ʻaʻole maopopo ka supernatant ma hope o ka centrifugation, hiki i ka paipu kecentrifuged no kekahi 5 min.
5. E hoʻonui i ka 0. 1 manawa i ka leo (10% o ka leo supernatant, ma kahi o 2.2 ml) o Endotoxin Scavenger i ka supernatant mai ka pae 4 a hoʻohuli i ka hui.E kau i ka hopena i loko o ka ʻauʻau hau a i ʻole e hoʻokomo ʻia i loko o ka hau hau (a i ʻole ka pahu hau hau) no 5 mau minuke a hiki i ka hoʻololi ʻana o ka hopena mai ka turbid a i ʻole a ʻaʻa (a i ʻole mau.ʻoluʻolu iki), a hui pū i kekahi manawa.
Δ Ma hope o ka hoʻohui ʻia ʻana o ka Endotoxin Scavenger i ka supernatant, e lilo ka supernatant i ʻūhū akāpono e lilo ka supernatant i akaka (a i ʻole ʻūlū iki paha) ma hope o ka hoʻoluʻu ʻana i ka ʻauʻau hau.
6. Ma hope o ka hoʻokomo ʻia ʻana o ka supernatant ma ka lumi wela (> 25 ℃) no 10 - 15 min, e lilo ia i ʻūlū e like mepiʻi ka mahana a hiki i ka lumi wela.A laila e hoʻohuli ʻia ka supernatant e hui.
Δ Inā haʻahaʻa ka mahana o ka lumi a makemake paha ʻoe e hōʻemi i ka manawa o ka unuhi ʻana, hiki ke hoʻokomo ʻia ka supernatant i loko o kahi ʻauʻau wai 37 ~ 42 ℃ no 5 - 10 min a hiki ke hoʻokō ʻia ka hana aʻe ma hope o ka supernatant.lilo i pohu.
7. E centrifuge i ka supernatant ma kahi o 11,000 rpm (12,000 × g) no 10 min ma ka lumi wela (pono ka mahana ma kahi o 25 ℃) e hoʻokaʻawale i ka pae.Aia ka DNA ma ka ʻaoʻao wai o luna aʻo ka papa ʻaila polū haʻahaʻa he endotoxin a me nā mea haumia ʻē aʻe.Hoʻololi i kaDNA-i loko o ka wai wai i kahi paipu hou ae hoolei i ka aila.
Δ Pono ka mahana i ka centrifugation ma luna o 25 ℃ no ka mea ʻaʻole hiki ke hoʻokaʻawale ʻia ka pae kūponohiki mai inā haʻahaʻa loa ka mahana.
Δ Inā ʻaʻole maikaʻi ka hoʻokaʻawale ʻana o ka pae, hiki ke hoʻololi ʻia ka mahana centrifugation i 30 ℃ ahiki ke hoʻonui i ka manawa o ka centrifugation i 15 min.
Δ Mai omo i ka ʻaila ʻaila polū no ka mea he endotoxin a me nā mea haumia ʻē aʻe.
Mechanism
Hoʻokuʻu ʻia ʻo Lysis Neutralization
◇ Hoʻohui i ka 7.5 ml Buffer P1
◇ Hoʻohui i ka 7.5 ml Buffer P2
◇ Hoʻohui i ka 7.5 ml Buffer P4
Ka wehe ʻana o Endotoxin
◇ E hoʻonui i 0. 1 manawa i ka nui supernatant o Endotoxin Scavenger
Nakinaki a holoi
◇ E hoʻonui i 0.5 manawa ka nui o ka isopropanol
◇ Hoʻohui i 10 ml Buffer PW
◇ Hoʻohui i 10 ml Buffer PW
Elution
◇ Hoʻohui i ka 1 – 2 ml Buffer TB a i ʻole Endotoxin-free ddH2O
