Buccal Swab / Ikarita ya FTA Ikarita ya ADN Yigunga Kit Genomic ADN Gukuramo cyangwa Igikoresho cyo Kweza muri Buccal Swabs
Ibisobanuro:
Sukura vuba ADN ya genomic yo mu rwego rwo hejuru ya buccal swab / Ikarita ya FTA.
◮Nta kwanduza RNase:Inkingi ya ADN Yonyine itangwa nigikoresho ituma bishoboka kuvana RNA muri ADN genomic utongeyeho RNase mugihe cyigeragezo, ikabuza laboratoire kwanduzwa na RNase idasanzwe.
◮Umuvuduko wihuse:Foregene Protease ifite ibikorwa byinshi kurenza protease isa, kandi igogora ingero za tissue vuba;igikorwa kiroroshye, kandi ibikorwa byo gukuramo ADN genomic birashobora kurangira muminota 20-80.
◮Byoroshye:Centrifugation ikorerwa mubushyuhe bwicyumba, kandi ntihakenewe 4 ° C ubushyuhe buke bwa centrifugation cyangwa imvura ya Ethanol ya ADN.
◮Umutekano:Nta gukuramo ibinyabuzima bisabwa.
◮Ubwiza bwo hejuru:ADN yakuweho ADN ifite ibice binini, nta RNA, nta RNase, hamwe na ion nkeya cyane, bishobora kuzuza ibisabwa mubushakashatsi butandukanye.
◮Sisitemu yo gukuraho Micro:Irashobora kongera ubunini bwa ADN ya genomic, ikaba yoroshye yo gutahura cyangwa kugerageza.
Ibisobanuro
Iki gikoresho gitanga uburyo bunoze kandi bwihuse bwo kubona ADN ya genomic yibanze cyane muri buccal swabs na Ikarita ya FTA (ibibara byamaraso).Gukoresha isosiyete yacu's idasanzwe ya ADN yonyine ya silika membrane izenguruka inkingi na formula, ifatanije na Foregene Protease, kwibanda cyane, ADN ya genomic yo mu rwego rwo hejuru irashobora gukurwa muminota 80.Inkingi yabugenewe idasanzwe yo kweza ihuza ADN genomic, kandi ADN irashobora gutorwa hamwe na bike (15μl) sisitemu yo gukuraho kugirango yongere ubunini bwa ADN ya genomic yabonetse, yorohewe no gutahura cyangwa kugerageza.Igikoresho kirashobora gutunganya icyitegererezo kimwe cyangwa byinshi icyarimwe, kandi inzira yo kweza ntisaba gukuramo ibintu kama nka fenol, chloroform, hamwe nigihe kinini cya isopropanol cyangwa imvura ya Ethanol, kandi imikorere iroroshye kandi itwara igihe.
Ibisobanuro
50 Imyiteguro
Ibigize ibikoresho
| Buffer ST1 |
| Buffer ST2 |
| Acrylamide |
| Buffer PW |
| Buffer WB |
| Buffer EB |
| Foregene Protease |
| ADN-Inkingi gusa |
| Amabwiriza |
Ibiranga ibyiza
-Nta kwanduza RNase: Inkingi ya ADN-Yonyine yatanzwe nigikoresho ituma bishoboka kuvana RNA muri ADN genomic utiriwe wongera RNase mugihe cyigeragezo, ukirinda laboratoire kwanduzwa na RNase idasanzwe.
-Umuvuduko wihuse: Foregene Protease ifite ibikorwa byinshi kuruta protease isa, igogora ingero vuba;imikorere yoroshye.
-Ibyoroshye: Centrifugation ikorwa mubushyuhe bwicyumba, kandi ntihakenewe 4 ° C ubushyuhe buke bwa centrifugation cyangwa imvura ya Ethanol ya ADN.
-Umutekano: Nta gukuramo reagent ikenewe.
-Ubuziranenge bwo hejuru: ADN yakuwe muri ADN ifite ibice binini, nta RNA, nta RNase, hamwe na ion nkeya cyane, bishobora kuzuza ibisabwa mubushakashatsi butandukanye.
-Micro-elution sisitemu: Irashobora kongera ubunini bwa ADN ya genomic, ikaba yorohereza gutahura cyangwa kugerageza.
Porogaramu
Irakwiriye kweza ADN genomic duhereye ku ngero zikurikira: swabs ya buccal, Ikarita ya FTA (irangi ryamaraso).
Ububiko hamwe nubuzima bwa Shelf
-Iki gikoresho gishobora kubikwa amezi 12 mugihe cyumye mubushyuhe bwicyumba (15-25 ° C);niba ikeneye kubikwa mugihe kirekire, irashobora kubikwa kuri 2-8 ° C.
Icyitonderwa: Niba bibitswe ku bushyuhe buke, igisubizo gikunda kugwa.Mbere yo gukoresha, menya neza ko ushira igisubizo mubikoresho mubushyuhe bwicyumba mugihe runaka.Nibiba ngombwa, shyushya mu bwogero bwa 37 ° C bwamazi muminota 10 kugirango ushongeshe imvura, hanyuma ubivange mbere yo kubikoresha.
-Foregene Protease igisubizo gifite formula idasanzwe, ikora iyo ibitswe mubushyuhe bwicyumba igihe kinini (amezi 3);ibikorwa byayo no gutuza bizaba byiza iyo bibitswe kuri 4 ° C, birasabwa rero kubibika kuri 4 ° C, ibuka kutabika kuri -20 ° C.
Inkingi yo kweza ifunze
Muri iki gikoresho, mugikorwa cyo gukuramo ADN genomic, inkingi yo kweza iramenyeshwa neza kuri sample enzymatique lysis ivanze nta ntambwe ya centrifugation, kandi inkingi yo kweza irashobora guhagarikwa kubera imisemburo ituzuye hamwe nubukonje bwinshi bwikitegererezo.
Impamvu zikurikira zishoboka nizi zikurikira:
1. Igogorwa ryuzuye ryimisemburo yintangarugero.
Icyifuzo: Igihe cyo gutunganya icyitegererezo cya Foregene Protease gishobora kongerwa muburyo bukwiye cyangwa ndengakamere gishobora gufatwa nyuma ya centrifugation saa 12,000 rpm (~ 13,400 × g) kuminota 5.
2. Gukoresha cyane ingero za tissue cyangwa tissue nini.
Icyifuzo: Nibyiza kutarenza 1 Buccal swab muri sample;niba icyitegererezo ari kinini cyane, ongera dosiye ya Buffer ST1, Protease ya Foregene, buffer ST2 ukurikije.
3. Icyitegererezo cyubwiza buri hejuru cyane.
Icyifuzo: Ingero zirashobora kuvangwa neza na mM 10 za Tris-HCl mbere yo gukuramo ADN genomic.
4. Ibice by'ikarita y'amaraso byanyoye.
Icyifuzo: Igihe cya centrifugation yinzibacyuho yintambwe ya 6 yumwanya wamaraso (Ikarita ya FTA) gukuramo genomic birashobora kwongerwa muburyo bukwiye.
Umusaruro muke cyangwa nta ADN
Hariho ibintu byinshi bigira ingaruka kumusaruro wa ADN genomic, harimo inkomoko yicyitegererezo, uburyo bwo kubika icyitegererezo, gutegura icyitegererezo, manipulation, nibindi.
ADN ya ADN ntishobora kuboneka mugihe cyo kuyikuramo
Impamvu zishoboka nizi zikurikira:
1. Kubika neza ingero cyangwa kubika igihe kirekire biganisha ku kwangirika kwa ADN.
Icyifuzo: Ibibyimba byo mu kanwa bigomba guhitamo icyitegererezo gishya, kandi ntabwo ari byiza gukoresha ibishishwa byabitswe mu bikorwa byo gukuramo ADN;icyitegererezo cyamaraso kigomba kwemeza ko ubuziranenge bujuje ibisabwa kandi igihe cyo kubika ntigikwiye kuba kirekire.
2. Gukoresha uduce duto cyane birashobora gutuma nta gukuramo ADN ikwiranye.
Icyifuzo: Kurikiza amabwiriza ya buccal swab yerekana amabwiriza yo kuyobora, hanyuma uhanagure inshuro nyinshi zishoboka kugirango selile zihagije zishobore kwomekwa kumanwa yo gukuramo ADN genomic;kubikuramo amaraso ntangarugero, ahantu haca amaraso harashobora kwiyongera muburyo bukwiye.
3. Foregene Protease ibitswe nabi, bigatuma igabanuka ryibikorwa byayo cyangwa kudakora.
Icyifuzo: Emeza uburyo bwo kubika Protease ya Foregene cyangwa uyisimbuze Protease nshya ya Foregene kugirango itangwe neza.
4. Kubungabunga bidakwiye ibikoresho cyangwa igihe cyo kubika ni birebire cyane, bikaviramo kunanirwa ibice bimwe mubikoresho.
Icyifuzo: Gura ibikoresho bishya bya Buccal swab ADN yo kwigunga kubikorwa bijyanye.
5. Buffer WB ntabwo yongeyeho etanol yuzuye.
Icyifuzo: Emeza ko buffer WB yongeraho ingano yukuri ya Ethanol yuzuye.
6. Eluent ntabwo yongewe neza muri firime ya silicone.
Icyifuzo: Ongeramo 65 ° C mbere yo gushyushya ibitonyanga bya eluent hagati ya membrane ya silicone hanyuma usige ubushyuhe bwicyumba kuminota 5 kugirango wongere imikorere ya elution.
ADN itanga umusaruro muke ADN yonyine
Impamvu zikurikira zishoboka nizi zikurikira:
1. Kubika neza ingero cyangwa kubika igihe kirekire biganisha ku kwangirika kwa ADN.
Icyifuzo: Guswera mu kanwa nibyiza ko byatoranijwe bishya, kandi ibishishwa byabitswe ntibigomba gukoreshwa mugukuramo ADN genomic.
2. Niba ingano yintangarugero ya tissue ari nto cyane, ibice bya ADN byakuweho bizaba bike.
Icyifuzo: Kurikiza amabwiriza yo gutoranya umunwa wo mu kanwa mu gitabo gikora, uhanagura inshuro nyinshi zishoboka kugirango selile zihagije zishobora kwomekwa kumanwa yo gukuramo ADN genomic.
3. Foregene Protease ibitswe nabi, bigatuma igabanuka ryibikorwa byayo cyangwa kudakora.
Icyifuzo: Emeza uburyo bwo kubika Protease ya Foregene cyangwa uyisimbuze Protease nshya ya Foregene kugirango itangwe neza.
4. Ibibazo bikomeye.
Icyifuzo: Koresha Buffer EB kugirango ukureho;niba ukoresha ddH2O cyangwa izindi ntore, wemeze ko pH ya eluate iri hagati ya 7.0-8.5.
5. Intore ntabwo yongeweho neza neza.
Icyifuzo: Ongeramo ibitonyanga byoroshye hagati ya silicone ya membrane hanyuma usige ubushyuhe bwicyumba kuminota 5 kugirango wongere imikorere ya elution.
6. Amazi ya elution yegeranya bike cyane.
Icyifuzo: Koresha eluent mugukuraho ADN genomic nkuko bisabwa mumabwiriza, byibuze munsi ya 15 μl.
Ubuziranenge buke bwa ADN genomic bwigunze
Ubushobozi buke bwa ADN bwa ADN bushobora gutera kunanirwa cyangwa ibisubizo bidashimishije byubushakashatsi bwo hasi, nka: enzymes ntishobora gucibwa, PCR ntishobora kubona agace ka gene yinyungu, nibindi.
Impamvu zishoboka nizi zikurikira:
1. Umwanda wa Heteroprotein, umwanda wa RNA.
Isesengura: Inkingi yo kweza ntabwo yogejwe hakoreshejwe Buffer PW;inkingi ya Buffer PW yoza isuku ntabwo yogejwe ikoresheje umuvuduko ukwiye wa centrifugal.
Icyifuzo: Menya neza ko nta mvura igwa muri supernatant mbere yo kongeramo Ethanol;menya neza koza inkingi yo kweza ukurikije amabwiriza, kandi iyi ntambwe ntishobora kuvaho.
2. Guhumanya ion.
Isesengura: Buffer WB yoza isuku inkingi yasibwe cyangwa yogejwe rimwe gusa, bivamo kwanduza ionic.
Icyifuzo: Witondere gukaraba Buffer WB inshuro 2 nkuko byateganijwe kugirango ukureho ion zisigaye bishoboka.
3. RNA yanduye.
Isesengura: RNase yamahanga yongewe kuri buffer;Buffer PW yo gukaraba ntiyari yo, bituma ibisigazwa bya RNase, bigira ingaruka kumikorere ya RNA yo hepfo, nko muri vitro transcription.
Icyifuzo: Foregene series nucleic aside yo kwigunga irashobora gukuraho RNA nta yongeyeho RNase, bityo buccal Swab / FTA Ikarita ya ADN Yigunga Kit ntigomba kongeramo RNase;witondere gukurikiza amabwiriza ya Buffer PW yoza inkingi, kandi iyi ntambwe ntishobora kuvaho.
4. Ibisigisigi bya Ethanol.
Isesengura: Buffer WB ntabwo yakoze ubusa ya centrifugation yubusa nyuma yo koza inkingi yo kweza.
Icyifuzo: Kora imikorere yubusa ya tube centrifugation ukurikije amabwiriza.
5. Ibindi byanduye.
Isesengura: Ingero zabitswe cyangwa ingero zidasanzwe ntiziteganijwe.
Icyifuzo: Witondere neza icyitegererezo nkuko byateganijwe.
