Gukuramo ADN Mini Kit
Iki gikoresho gikoresha sisitemu ya buffer nziza hamwe na silika gel inkingi yo gutunganya, ishobora kugarura ibice 70 bp - 20 kb ADN ibice bitandukanye bya TAE cyangwa TBE agarose gel.Inkingi ya ADN ya adsorption irashobora kwamamaza ADN muburyo bwumunyu mwinshi.Byongeye kandi, igikoresho gishobora kweza mu buryo butaziguye ibice bya ADN biva mu bicuruzwa bya PCR, sisitemu yo gufata ibintu mu buryo bwa enzymatique cyangwa ibicuruzwa bya ADN bitavanze byabonetse mu bundi buryo, kandi bigakuraho umwanda nka poroteyine, ibindi bintu kama kama, iyoni y’umunyu ngugu na oligonucleotide primers.Irashobora kwemeza ko kwezwa gushobora kurangira muminota 10-15min.ADN yatunganijwe irashobora gukoreshwa muburyo butaziguye, guhinduka, guhinduranya enzyme, muri vitro transcription, PCR, ikurikiranye, microinjection, nibindi.
Imiterere yo kubika
Ubike kuri -15 ~ -25 ℃ no gutwara ubushyuhe bwicyumba.
Ibigize
| Ibigize | (100 rxns) |
| Buffer GDP | 80 ml |
| Buffer GW | 2 × 20 ml |
| Buffer | 20 ml |
| Byihuta bya ADN Mini Inkingi-G | 100 |
Buffer GDP:ADN ihuza buffer.
Buffer GW:Gukaraba;ongeramo etanol yuzuye nubunini bwerekanwe kumacupa mbere yo gukoresha.
Buffer Elution:Gukuraho.
Inkingi ya ADN Yihuta-G:Inkingi ya ADN.
Igikoresho cyo gukusanya 2 ml:Gukusanya imiyoboro yo kuyungurura.
Ibikoresho Byateguwe
1.5 ml ya sterisile tebes, etanol yuzuye na isopropanol (mugihe agace ka ADN ≤100 bp, ongeramo umuzingo 1
isopropanol kugeza kuri 1 jel), kwiyuhagira amazi.
Inzira y'Ubushakashatsi
Ongeramo ml 80 ya Ethanol kugirango ugabanye Buffer GW nkuko bigaragara kuri tagi mbere yo kuyikoresha, ubike ubushyuhe bwicyumba.
Urwego
1. Igisubizo cya PCR
Gahunda yo gukuramo Gel: Ongeraho ingano ingana Buffer GDP PCR igisubizo cyo kugarura igisubizo:Ongeramo inshuro 5 ingano Buffer
2. GDP Kubara ingano ya gel (100) μbingana na 100 mg)
Kuramo gel
3. Shyushya kuri 50 ~ 55℃
4. Gukaraba
Ongeramo 300 μL ya Buffer GDP *
Ongeramo 700 μL ya Buffer GW
Ongeramo 700 μL ya Buffer GW
5. Elute
Ongeramo 20 - 30μL ya Elution Buffer cyangwa amazi ya deionised
Icyitonderwa * PCR reaction ya fluid isubirana nta ntambwe
Gahunda yo gukuramo Gel
1. Nyuma ya ADN ya electrophoreis kugirango igabanye ibice bya ADN, shishoza umurongo umwe wibice bya ADN bivuye muri gel ya agarose munsi yumucyo UV.Birasabwa gukoresha impapuro zikurura kugirango ushiremo ububobere bugaragara bwa gel kandi ugabanye ubunini bwigice cya gel ukuraho agarose yinyongera bishoboka.Gupima ibice bya gel (udafite microcentrifuge) kugirango ubare ingano yacyo: Ingano ya 100 mg gelslice igera kuri 100 μL, ukeka ko ubucucike ari 1g / ml.
2. Ongeramo ingano ingana na Buffer GDP, incubate kuri 50 ~ 55 ℃ muminota 7-10 (ukurikije ubunini bwa gel, hindura igihe cyo gukora kugeza igihe geli ishonga burundu).Hindura umuyoboro inshuro 2 mugihe cya incubation.
Ongeraho umubumbe wa 1-3 wa Buffer GDP ntabwo bizahindura imikorere ya ADN.Niba agace ka ADN kagaruwe <100 bp, hagomba kongerwaho umubumbe wa 3 wa Buffer GDP;mugihe ibice bya gel bimaze gushonga burundu, ongeramo inomero 1 ya isopropanol hanyuma uvange neza, hanyuma ukomeze kumuntambwe ikurikira.
3. Centrifuge muri make kugirango uzane icyitegererezo munsi yigituba, shyiramo Mini Mini Inkingi ya ADP yihuta-G muri Tube ya 2 ml, wohereze witonze igisubizo kinini cya 700 μL rimwe a
igihe cyo kuyungurura inkingi, centrifuge kuri 12,000 rpm (13.800 X g) kumasegonda 30-60.
4. Hagarika akayunguruzo hanyuma wongereho 300 μL ya Buffer GDP kuri inkingi, ubike ubushyuhe bwicyumba kuminota 1, centrifuge kuri 12,000 rpm (13.800 X g) kumasegonda 30-60.
5. Hagarika akayunguruzo hanyuma wongereho 700 μL ya Buffer GW (reba niba Ethanol yuzuye yongewe mbere!) Kurinkingi, centrifuge saa 12,000 rpm (13.800 X g) kumasegonda 30-60.
Δ Nyamuneka ongeramo Buffer GW kuzengurutse urukuta rwa adsorption, cyangwa wongereho Buffer GW igifuniko cy'inyuma hanyuma ubivange hejuru inshuro 2 - 3 kugirango ufashe guhanagura rwose umunyu wiziritse kurukuta.
6. Subiramo intambwe ya 5.
Guswera hamwe na Buffer GW inshuro ebyiri birashobora kwemeza ko umunyu ukuweho burundu kandi bigakuraho ingaruka kubushakashatsi bwakurikiyeho.
7. Hagarika filtrate na centrifuge inkingi yubusa kuri 12.000 rpm (13.800 X g) kuminota 2.
8. Shyiramo inkingi mumazi ya 1.5 ya microcentrifuge isukuye, ongeramo 20 - 30 μL ya Elution Buffer hagati yinkingi ya membrane, incubate muminota 2, hanyuma centrifuge kuri 12.000 rpm (13.800 X g) kuminota 1.Hagarika inkingi, ubike ADN wabonye kuri -20.
Kwimura ndengakamere yintambwe ya 8 kumurongo kugirango wongere utere hejuru kandi ushushe Elution Buffer kuri 55 (mugihe agace ka ADN> 3 kb) birashoboka ko byafasha kongera imikorere yo gukira.
Porogaramu yo kugarura ibicuruzwa bya PCR
Iyi protocole irakoreshwa mugusukura ibice bya ADN biva mubicuruzwa bya PCR, sisitemu ya reaction enzymatique nibindi bicuruzwa bya ADN bitavanze (harimo na ADN genetique).Iki gisubizo kirashobora gukuraho neza nucleotide zitandukanye, primers, primer dimers, molekile yumunyu, enzymes nibindi byanduye.
1. Muri make centrifuge ibicuruzwa bya PCR, igisubizo cya enzymatique reaction, nibindi bicuruzwa bya ADN bitavanze.Gereranya ingano yabyo hamwe na pipette hanyuma wohereze kuri 1.5 sterile sterile cyangwa ml 2.Ongeramo ddH2O kugeza amajwi agera kuri 100 μL;mugihe kuri ADN genomic ifite ubwumvikane buke, kuvanga kugeza kuri 300 μL hamwe na ddH2O bizafasha kunoza imikorere yo gukira.
2. Ongeramo umubumbe wa 5 wa Buffer GDP, vanga neza ukoresheje inverting cyangwa vortexing.Niba ADN igice cyinyungu> 100 bp, hiyongereyeho umubumbe wa 1.5 (sample + Buffer GDP) ya Ethanol.
3. Ongera inkingi usubire mu muyoboro wo gukusanya, ohereza imvange ku nkingi, centrifuge kuri 12.000 rpm (13.800 × g) kuri 30 - 60 sec.Niba ingano yumuti uvanze ari> 700 µL, shyira inkingi ya adsorption mumurongo wo gukusanya, ohereza igisubizo gisigaye kumurongo wa adsorption, na centrifuge kuri 12.000 rpm (13.800 × g) kumasegonda 30 - 60.
4. Imikorere ikurikira yerekeza ku ntambwe 5 - 8 ya 08- 1 / Gahunda yo gukuramo Gel.
Porogaramu
Ubwinshi butandukanye bwa TAE cyangwa TBE agarose gel;Ibicuruzwa bya PCR, sisitemu ya reaction ya enzymatique cyangwa ibindi bicuruzwa bya ADN bitavanze byabonetse muburyo butandukanye.Ibice byagarutsweho byatandukanijwe kuva70 bp -20 kb.
Inyandiko
Kubushakashatsi ukoreshe gusa.Ntabwo ari ugukoresha muburyo bwo gusuzuma.
1. Ongeramo ml 80 ya Ethanol kugirango ugabanye Buffer GW nkuko bigaragara kuri tagi mbere yo kuyikoresha, ubike ubushyuhe bwicyumba.
2. Niba Buffer GDP yoroshye kugwa mugihe cyo kubika ubushyuhe buke, irashobora gushyirwa mubushyuhe bwicyumba mugihe runaka mbere yo kuyikoresha.Bibaye ngombwa, irashobora gushyukwa muri 37 bath ubwogero bwamazi kugeza imvura iguye burundu, hanyuma irashobora gukoreshwa nyuma yo kuvanga.
3. Shyira ubushyuhe bwamazi kuri 50 ~ 55 ℃ mbere.
4. Muri gahunda yo gukuramo 08-1 / Gel intambwe ya 1, kugabanya ingano ya gice ya gel bizagabanya cyane igihe cyo gushonga kandi byongere imikorere yo gukira (ADN Linearized ADN byoroshye hydrolyze mugihe ikomeje kugaragara mubushyuhe bwinshi).Ntugaragaze gel ya ADN muri UV igihe kirekire, kuko urumuri ultraviolet rushobora kwangiza ADN.
5. Gabanya gele muri 08- 1 / Gahunda yo gukuramo Gel intambwe ya 2 burundu, bitabaye ibyo imikorere ya ADN yo gukira izagira ingaruka zikomeye.
6. Shyushya Buffer cyangwa ddH2O kugeza 55 ℃, ifasha kunoza imikorere ya ADN.Birasabwa kubika ADN mu buryo bwa mM 2,5 Tris-HCl, pH 7.0 - 8.5.
