EndoFree Plasmid Maxi Kit
Le khithi ifanelekile ukutsalwa kwi-150 - 300 ml yesisombululo sebhaktheriya ekhuliswe ubusuku bonke, usebenzisa indlela ephuculweyo ye-SDS-alkaline lysis ukuze i-lyse ibhaktheriya.Isicatshulwa esikrwada sidityaniswe ngokukhethiweyo kunye ne-Endotoxin Scavenger ekhethekileyo kwaye yahlulwe yi-centrifugation ukususa i-endotoxins.Emva koko, i-silica gel membrane ibophelela ngokukhethekileyo kwi-plasmid DNA kwisisombululo phantsi kweemeko zetyuwa ephezulu kunye ne-pH ephantsi.Oku kulandelwa kukongezwa kwesithinteli sokuhlamba ukususa ubumdaka kunye nezinye iinxalenye zebhaktiriya.Okokugqibela, ityuwa ephantsi, i-high-pH elution buffer isetyenziselwa ukukhupha i-plasmid esulungekileyo ye-DNA esuka kwi-silicon matrix membrane.I-silica gel membrane isebenzisa inwebu ekhethekileyo ye-adsorption, kwaye i-adsorption yemali umahluko phakathi kwekholomu kunye nekholomu incinci kakhulu kwaye ukuphindaphinda kulungile.I-Phenol, i-chloroform kunye nezinye ii-reagents ezinetyhefu azifuneki, kwaye akukho manyathelo e-ethanol emvula.Le kit ingasetyenziselwa ukukhupha ngokukhawuleza i-0.2 -1.5 mg ye-DNA ye-plasmid ecocekileyo ephezulu, kunye nesantya sokutsalwa kwe-80% -90%.Ikiti isebenzisa ifomula yenkqubo ekhethekileyo isusa i-endotoxin, umxholo we-endotoxin uphantsi kakhulu kwaye umphumo wokudluliselwa kweseli ugqwesileyo.I-plasmid ekhutshiweyo ingasetyenziswa ngokuthe ngqo kwi-enzyme digestion, i-PCR, i-in vitro transcription, ukuguqulwa, ulandelelwano kunye nezinye iimvavanyo zebhayoloji ye-molekyuli.
Iimeko zokugcina
I-RNaseA kufuneka igcinwe ku -30 ~ -15℃ kwaye ihanjiswe ku-≤0℃.
I-Endotoxin Scavenger inokugcinwa kwi-2 ~ 8 ℃ inyanga enye, igcinwe kwi -30 ~ -15 ℃ yokugcina ixesha elide.kwaye ithuthwe nge-≤0℃.
Amanye amacandelo kufuneka agcinwe kwiqondo lobushushu begumbi (15 ~ 25℃) kwaye ahanjiswe kwiqondo lobushushu begumbi.
Amacandelo
| Amacandelo | 10RXNS |
| RNase A | 750 μL |
| Isithinteli P1 | 75 ml |
| Isithinteli P2 | 75 ml |
| Isithinteli P4 | 75 ml |
| Endotoxin Scavenger | 25 ml |
| Isithinteli PW | 2 × 22 ml |
| Isithinteli TB | 20 ml |
| I-FastPure DNA Maxi Columns ( Nganye kwi-50ml Collection Tube) | 10 |
| I-Endotoxin-free Collection Tube | 2 × 5 |
RNaseA:I-10 mg / ml, esetyenziselwa ukususa i-RNA.
Isithinteli P1:isithinteli sokunqunyanyiswa kwebhaktiriya, yongeza i-RNaseA kwi-Buffer P1 ngaphambi kokusetyenziswa kokuqala.
Isithinteli P2:ibhaktheriya lysis buffer (equlathe SDS/NaOH).
Isithinteli P4:isithinteli esiphazamisayo.
I-Endotoxin Scavenger:susa ngokufanelekileyo i-endotoxin kwisicatshulwa seplasmid ekrwada.
Isithinteli PW:hlamba isithinteli, yongeza umthamo ochaziweyo we-ethanol ngaphambi kokuba uyisebenzise okokuqala.
Isithinteli TB:lution buffer.
I-FastPure DNA Maxi Columns:iikholamu ze-plasmid DNA adsorption.
Imibhobho yoQokelelo 50 ml:iityhubhu zokuqokelela ukuhluza.
I-Endotoxin-free Collection Tube:iityhubhu zokuqokelelwa kwe-plasmid DNA.
Izinto Ezilungisiweyo
I-ethanol epheleleyo, i-isopropanol, i-50 ml yetyhubhu ye-centrifuge engqukuva kunye ne-50 ml ye-endotoxin-freeiityhubhu ze-centrifuge.
Usetyenziso
Le mveliso ilungele ukutsalwa okukhulu kweeplasmids ukusuka kwi-150 - 300 ml yesisombululo sebhaktheriya.ikhuliswe ngobusuku.
Inkqubo yoVavanyo
1. Thatha i-150 – 200 ml (akukho ngaphezulu kwe-300 ml) yesisombululo sebhaktiriya esikhuliswe ngobusuku kunye ne-centrifugemalunga ne-11,000 rpm (12,000 × g) kwi-1 - 2 min.Lahla i-supernatant kwaye uqokelele ibhaktheriya.
Δ Xa uqokelela ngaphezu kwe-50 ml yesisombululo sebhaktheriya, ibhaktheriya inokuqokelelwa ngokudibanisa isisombululo sebhaktheriya, i-centrifugation, ukulahla i-supernatant kunye namanye amanyathelo kwityhubhu enye ye-50 ml.
amaxesha amaninzi.
2. Yongeza i-7.5 ml ye-Buffer P1 (nceda ujonge ukuba i-RNaseA yongezwe kwi-Buffer P1) kwi-centrifugeityhubhu equkethe ibhaktheriya kwaye ixube ngokucokisekileyo nge-vortex okanye ipayipi.
Δ Ukumiswa ngokupheleleyo kweebhaktheriya kule nyathelo kubalulekile ekuveliseni, kwaye akufanele kubekho i-bacterial clumps emva kokumiswa.Ukuba kukho i-bacterial clumps engaxutywanga ngokucokisekileyo, iya kuchaphazela i-lysis, ibangele isivuno esiphantsi kunye nobunyulu.Ukuba i-OD600 yesisombululo sebhaktheriya ngu-0.65, kucetyiswa ukuba i-7.5 ml ye-Buffer P1 isetyenziswe xa ikhupha kwi-150 ml yesisombululo sebhaktheriya;xa i-OD600 ingu-0.75, i-8 ml ye-Buffer P1 kufuneka isetyenziswe kwaye imiqulu ye-Buffers P2 kunye ne-P4 kufuneka itshintshwe ngokufanelekileyo.Ukuba umthamo wesisombululo sebhaktheriya unyuswe ukuya kuma-200 ml, kucetyiswa ukubaumthamo we-Buffers P1, P2, kunye ne-P4 zonyuswe ngokomlinganiselo.
3. Yongeza i-7.5 ml ye-Buffer P2 ekumisweni kwebhaktiriya ukusuka kwinqanaba lesi-2 kwaye udibanise ngobunono phezulu naphantsi kwi-6 - 8.amaxesha kwaye udibanise kwiqondo lokushisa kwe-4 - 5 min.
Δ Guqula ngobunono ukuze udibanise ngokucokisekileyo.I-Vortexing iya kubangela ukuhlukana kwe-DNA ye-genomic, okukhokelela kwiinqununu ze-DNA ze-genomic kwi-plasmid ekhutshwe.Ngeli xesha, isisombululo siba yi-viscous kunye ne-translucent, ebonisa ukuba ibhaktheriya i-lysed ngokupheleleyo.Ubude bexesha akufanele budlule i-5 min ukuphepha ukutshatyalaliswa kweeplasmids.Ukuba isisombululo asicacanga, kunokubakho iibhaktheriya ezininzi ezibangelai-lysis engaphelelanga, ngoko ke inani lebhaktheriya kufuneka lincitshiswe ngokufanelekileyo.
4. Yongeza i-7.5 ml ye-Buffer P4 ekumisweni kwebhaktheriya ukusuka kwinqanaba lesi-3 kwaye ngokukhawuleza ujike ngokukhawuleza 6 - 8 amaxesha ukuvumela isisombululo ukuba sinciphise ngokupheleleyo i-Buffer P2.Ngeli xesha, i-white flocculent precipitate kufuneka ibonakale.I-Centrifuge ngaphezu kwe-11,000 rpm (12,000 × g) ye-10 - 15 min, i-pippette ngononophelo i-supernatant ibe ityhubhu entsha ye-50 ml engqukuva-phantsi (ezilungiseleleyo), kwaye uphephe.nqwenela imvula edadayo emhlophe.
Δ Yongeza i-Buffer P4 kwaye ngokukhawuleza uguqule ukuxuba kakuhle.Shiya ityhubhu ukuba ime kude kube yilapho i-precipitate emhlophe isasazwa ngokulinganayo kwisisombululo sokuthintela ukuveliswa kwemvula yendawo enokuthi ichaphazele ukungathathi hlangothi.Ukuba akukho flocculent iyunifomu emhlophe phambi kwe-centrifugation kwaye i-supernatant ayicacanga emva kwe-centrifugation, ityhubhu ingabai-centrifuged enye i-5 min.
5. Yongeza i-0. 1 amaxesha omthamo (i-10% yomthamo omkhulu, malunga ne-2.2 ml) ye-Endotoxin Scavenger ukuya kwi-supernatant ukusuka kwinqanaba le-4 kwaye uguqule ukuxuba.Beka isisombululo kwibhafu yomkhenkce okanye usifake kumkhenkce otyumkileyo (okanye kwisikhenkcezisi) kangangemizuzu emi-5 de isisombululo sitshintshe ukusuka kwi-turbid ukuya ekucaceni nasekukhanyeni (okanye sisahleli.i-turbid encinci), kwaye ngamanye amaxesha udibanise amaxesha amaninzi.
Δ Emva kokuba i-Endotoxin Scavenger yongezwe kwi-supernatant, i-supernatant iya kuba sisidubedube kodwaI-supernatant kufuneka icace (okanye i-turbid kancinane) emva kokupholisa kwibhafu yomkhenkce.
6. Emva kokuba i-supernatant ibekwe kwiqondo lobushushu legumbi (>25℃) kangange-10 – 15 min, iya kuba sisidubedube njengoko.ubushushu bayo buyanda ukuya kwiqondo lobushushu begumbi.Emva koko i-supernatant kufuneka iguqulwe ukuze ixube.
Δ Ukuba ubushushu begumbi busezantsi okanye ufuna ukunciphisa ixesha lokukhutshwa, i-supernatant inokufakwa kwi-37 ~ 42 ℃ yokuhlambela amanzi kwi-5 - 10 min kwaye inyathelo elilandelayo linokuqhutywa emva kwe-supernatant.iba sisidubedube.
7. I-Centrifuge i-supernatant malunga ne-11,000 rpm (12,000 × g) ngemizuzu eyi-10 kwiqondo lobushushu begumbi (iqondo lobushushu kufuneka libe>>25℃) ukwahlula isigaba.Isigaba esiphezulu se-aqueous siqulethe i-DNA ngelixa i-low blue phase layer ine-endotoxin kunye nezinye izinto ezingcolileyo.Dlulisa iI-DNA-equlethe isigaba samanzi kwi-tube entsha kunyelahla umaleko onamafutha.
Δ Iqondo lobushushu ngexesha le-centrifugation kufuneka libe ngaphezu kwe-25℃ njengoko ulwahlulo lwesigaba olusebenzayo lungenzikwenzeka ukuba ubushushu buphantsi kakhulu.
Δ Ukuba ulwahlulo lwesigaba alusebenzi, iqondo lobushushu le-centrifugation lingahlengahlengiswa libe yi-30℃ kwayeixesha le-centrifugation linganyuswa ukuya kwi-15 min.
Δ Sukumunca umaleko weoyile oluhlaza njengoko uqulethe i-endotoxin kunye nobunye ukungcola.
Inkqubo
Ukumiswa kwakhona kweLysis Neutralization
◇ Yongeza 7.5 ml Isithinteli P1
◇ Yongeza 7.5 ml Isithinteli P2
◇ Yongeza 7.5 ml Isithinteli P4
Ukususwa kwe-endotoxin
◇Yongeza ngo-0. 1 umphinda-phinde ngomthamo omkhulu we-Endotoxin Scavenger
Ukubophelela kunye nokuHlanjwa
◇ Yongeza ngokuphindwe ka-0.5 umthamo we-isopropanol
◇ Yongeza 10 ml Isithinteli PW
◇ Yongeza 10 ml Isithinteli PW
Uvavanyo
◇ Yongeza i-1 – 2 ml ye-Buffer TB okanye i-Endotoxin-free ddH2O
