EndoFree Plasmid Maxi Kit
He pai tenei kete mo te tangohanga mai i te 150 – 300 ml o te wairewa huakita i whakatipuhia mo te po, ma te whakamahi i te tikanga lysis SDS-waikawa pai ake ki te lyse i nga huakita.Ko te tangohanga masivesivá kua kowhiria me te Endotoxin Scavenger ahurei ka wehea e te centrifugation hei tango endotoxins.Na, ka herea te kirikiri kirikiri ki te DNA plasmid i roto i te otinga i raro i nga tikanga o te tote teitei me te pH iti.Whai muri i tenei ko te taapiri i te parapara horoi hei tango i nga poke me etahi atu waahanga huakita.Ka mutu, ka whakamahia te tote iti, te teitei-pH parapara elution ki te whakakore i te DNA plasmid parakore mai i te membrane matrix silicon.Ka whakamahia e te kiriuhi kirikiri kirikiri he kirikiri adsorption motuhake, a he iti rawa te rereketanga o te nui o te adsorption i waenga i te poupou me te poupou, he pai hoki te tukurua.Ko te Phenol, te chloroform me etahi atu reagents paitini kaore e hiahiatia, kaore ano hoki nga hikoi rerenga waihano.Ka taea te whakamahi i tenei kete ki te tango tere i te 0.2 -1.5 mg o te DNA plasmid plasmid parakore teitei, me te tere tangohanga o te 80% -90%.Ka whakamahia e te kete he tauira tikanga motuhake hei tango i te endotoxin, he iti rawa te ihirangi o te endotoxin me te pai o te awenga whakawhiti pūtau.Ka taea te whakamahi tika i te plasmid kua tangohia ki roto i te nakunaku whākōkī, PCR, i roto i te whakamaoritanga in vitro, te hurihanga, te raupapa me etahi atu whakamatautau koiora irapoi.
Nga tikanga rokiroki
Me penapena te RNaseA ki te -30 ~ -15 ℃ ka haria ki te ≤0 ℃ .
Ka taea te penapena Endotoxin Scavenger ki te 2 ~ 8 ℃ mo te marama kotahi, ka rongoa ki te -30 ~ -15 ℃ mo te rokiroki mo te wa roa.ka kawea ki te ≤0 ℃.
Ko etahi atu waahanga me penapena ki te pāmahana rūma (15 ~ 25 ℃) ka haria ki te pāmahana rūma.
Waehanga
| Waehanga | 10RXNS |
| RNase A | 750 μL |
| Kaipupuri P1 | 75 ml |
| Kaipupuri P2 | 75 ml |
| Pūreirei P4 | 75 ml |
| Endotoxin Scavenger | 25 ml |
| Kaipupuri PW | 2 × 22 ml |
| Pupuri TB | 20 ml |
| Nga Tiwae Maxi DNA Pure Tere ( Ia i roto i te Tube Kohinga 50ml) | 10 |
| Ngongo Kohinga Endotoxin-kore | 2 × 5 |
RNaseA:10 mg/ml, whakamahia hei tango i te RNA.
Pūrei P1:parepare whakatārewatanga huakita, tāpirihia te RNaseA ki te Buffer P1 i mua i te whakamahi tuatahi.
Pūrei P2:parepare lysis huakita (kei roto SDS/NaOH).
Pūrei P4:parepare whakakore.
Whakamutunga Endotoxin:te tango i te endotoxin mai i te tangohanga plasmid masivesivá.
Pupuri PW:horoi i te parapara, tāpirihia te rōrahi o te waihā i mua i te whakamahinga tuatahi.
Pupuri TB:pūreirei hauhā.
Nga Tiwae Maxi DNA Pure Tere:plasmid DNA adsorption tīwae.
Ngongo Kohinga 50 ml:ngongo kohinga filtrate.
Ngongo Kohinga Endotoxin-kore:ngongo kohinga DNA plasmid.
Rauemi Kua Whakaritea
Ewaro tino, isopropanol, 50 ml ngongo centrifuge porotaka-raro me te 50 ml endotoxin-korengongo centrifuge.
Nga tono
He pai tenei hua mo te tangohanga nui o nga plasmids mai i te 150 - 300 ml o te otinga huakita.i mahia mo te po.
Tukanga Whakamatau
1. Tangohia te 150 – 200 ml (kaua e neke ake i te 300 ml) o te wairewa huakita kua whakatipuria mo te po me te centrifuge iāhua 11,000 rpm (12,000 × g) mō te 1 – 2 min.Makahia te supernatant ka kohikohi huakita.
Δ I te kohikohi neke atu i te 50 ml o te wairewa huakita, ka taea te kohia nga huakita ma te taapiri i te otinga huakita, te centrifugation, te whakakore i te supernatant me etahi atu taahiraa i roto i te ngongo 50 ml kotahi mo
maha nga wa.
2. Taapirihia te 7.5 ml o te Buffer P1 (tirohia mehemea kua tapirihia te RNaseA ki te Buffer P1) ki te centrifugengongo kei roto i te huakita me te uru pai ma te vortex me te paipa.
Δ Ko te whakatārewatanga katoa o te huakita i roto i tenei taahiraa he mea tino nui ki te tuku, a kia kaua he putunga huakita i muri i te whakatārewatanga.Mena he kapoi huakita kaore i tino whakaranuhia, ka pa ki te lysis, ka iti te hua me te ma.Mena ko te OD600 o te otinga huakita he 0.65, e taunaki ana kia whakamahia te 7.5 ml o te Buffer P1 ina tangohia mai i te 150 ml o te otinga huakita;ina he 0.75 te OD600, me whakamahi te 8 ml o te Buffer P1 me te whakarereke i nga pukapuka o te Buffers P2 me te P4.Mena ka whakanuia te rōrahi o te otinga huakita ki te 200 ml, ka tūtohu kiaKo te rahinga o nga Kaipupuri P1, P2, me te P4 ka whakanuia kia riterite.
3. Taapirihia te 7.5 ml o te Buffer P2 ki te whakatārewatanga huakita mai i te taahiraa 2 ka uru marie ki runga me raro mo te 6 – 8nga wa ka whakakororia i te pāmahana rūma mo te 4 – 5 min.
Δ Huri ata kia uru pai.Ma te Vortexing ka pakaru te DNA ira tangata, ka puta mai he maramara DNA ira i roto i te plasmid kua tangohia.I tenei wa, ka pokarekare te wairewa, ka marama, e whakaatu ana kua tino lysed nga huakita.Ko te roanga kia kaua e neke ake i te 5 meneti hei karo i te whakangaromanga o nga plasmids.Ki te kore e marama te otinga, he nui rawa pea nga huakita ka putakare i oti te lysis, no reira me whakaheke tika te nui o nga huakita.
4. Tāpirihia te 7.5 ml o te Pūreira P4 ki te whakatārewatanga huakita mai i te taahiraa 3 ka huri marietia kia 6 – 8 nga wa kia taea ai e te otinga te whakakore i te Buffer P2.I tenei wa, ka puta mai he rerenga ma.Ko te Centrifuge neke atu i te 11,000 rpm (12,000 × g) mo te 10 – 15 min, ka ata pippeti te supernatant ki roto i te ngongo centrifuge hou 50 ml huri noa-raro (kua rite), ka karoaspirate te parapara ma e rere ana.
Δ Taapirihia te Buffer P4 ka huri tonu kia uru pai.Waiho te ngongo kia tu tonu kia tohatoha noa te rerenga ma puta noa i te otinga hei aukati i te whakaputanga o te rerenga o te rohe ka pa ki te aukati.Mena karekau he rewharewha ma i mua i te centrifugation me te kore e marama te supernatant i muri i te centrifugation, ka taea te ngongocentrifuged mo tetahi atu 5 min.
5. Tāpirihia te 0. 1 whakarea te rōrahi (10% o te rōrahi supernatant, tata ki te 2.2 ml) o Endotoxin Scavenger ki te supernatant mai i te taahiraa 4 ka huri kia uru.Whakanohoia te otinga ki roto i te kaukau tio, whakauruhia ranei ki roto i te huka tio kuru (te pouaka pouaka pouaka pouaka ranei) mo te 5 meneti kia huri te otinga mai i te purupuru ki te maamaa me te marama (he tonu ranei.paku kerekere), me te whakaranu i etahi wa maha.
Δ I muri i te whakaurunga o te Endotoxin Scavenger ki te supernatant, ka pouri te supernatant engarime marama (he pakupaku ranei) te supernatant i muri i te whakamatao i roto i te pati hukapapa.
6. I muri i te tuunga o te supernatant ki te pāmahana rūma (> 25 ℃) mo te 10 – 15 min, ka pourika piki tona pāmahana ki te pāmahana rūma.Na me huri te supernatant ki te uru.
Δ Mena he iti ake te pāmahana o te rūma, kei te pirangi ranei koe ki te whakaiti i te wa tangohanga, ka taea te whakawhao i te supernatant ki roto i te kaukau wai 37 ~ 42 ℃ mo te 5 - 10 min ka taea te mahi i muri mai i te supernatant.ka kopouri.
7. Whakanuia te supernatant i te 11,000 rpm (12,000 × g) mo te 10 min i te pāmahana rūma (me 25 ℃ te pāmahana) hei wehe i te wahanga.Kei roto i te wahanga wai o runga te DNA, ko te paparanga hinuhinu puru o raro kei roto te endotoxin me etahi atu poke.Whakawhiti teDNA-kei roto te wahanga wai ki te ngongo hou me tewhakarerea te paparanga hinu.
Δ Ko te pāmahana i roto i te centrifugation me neke ake i te 25 ℃ na te mea karekau te wehenga o te wahanga whai huaka puta mena he iti rawa te pāmahana.
Δ Mena kaore i te whai hua te wehenga o te waahanga, ka taea te whakatika i te pāmahana centrifugation ki te 30 ℃ meka taea te whakanui i te wa o te centrifugation ki te 15 min.
Δ Kaua e ngotea te paparanga hinu kikorangi na te mea kei roto te endotoxin me etahi atu poke.
Hangaia
Resuspension Lysis Neutralization
◇ Tāpirihia te 7.5 ml Pūrei P1
◇ Tāpirihia te 7.5 ml Pūrei P2
◇ Tāpirihia te 7.5 ml Buffer P4
Te tango endotoxin
◇ Tāpirihia te 0. 1 whakareatanga te rōrahi pūmua o Endotoxin Scavenger
Te here me te horoi
◇ Tāpirihia kia 0.5 whakareatanga te rōrahi o te isopropanol
◇ Tāpirihia te 10 ml Buffer PW
◇ Tāpirihia te 10 ml Buffer PW
Whakawhanawhana
◇ Tāpirihia te 1 – 2 ml Buffer TB or Endotoxin-free ddH2O
