Inama zo kunoza uburyo bwo kugarura kole

1. Ongera umutwaro wikitegererezo mugihe cya electrophoreis.

2. Koresha buffer ya electrophoreis nshya.

3. Mugihe ukata kole, gerageza gukata kole gusa ukoresheje imirongo kugirango ugabanye ingano yo gukata kole: ntukeneye kole hamwe nibice bike bigamije, bitabaye ibyo bizagira ingaruka kumuvuduko wo gukira.

4. Nyuma yo gushonga ibice bibiri cyangwa byinshi bya kole, koresha umuyoboro nubwo ingano yaba ingana gute hanyuma uyohereze kumurongo umwe.

5. Igisubizo cyongewe muri sol kirashobora kuba gito cyane, kikaba gifasha cyane guhuza ADN muri membrane, ariko muri rusange ntibirenza 750ul.

6. Urufunguzo rwo gukira gel ni uguhuza ADN inkingi binyuze mumunyu mwinshi, acide (charge) hamwe na hydrophobicite yumuti murinkingi.Kubwibyo, niba pH ya buffer ya electrophoreis iri hejuru cyane, 10ul (pH 5.0, 3mol / L NaAC) irashobora kongerwaho sol;murwego rwo kurushaho gufata molekile ya ADN kuri membrane, 30% isopropanol irashobora kongerwamo ubushyuhe mumazi nyuma yo gushonga kole.

7. Mbere yo kongeramo eluent, usige inkingi mubushyuhe bwicyumba muminota mike (hafi iminota 10) kugirango uhumeke neza Ethanol.

8. Hanyuma, ongeramo bike kugirango ugabanye ingano yo kugarura.Mubisanzwe, 30-50μl eluent ikoreshwa mugukuraho (ntabwo ari bike cyane, bitabaye ibyo ntabwo izashobora guhanagura membrane, idafasha kurandura);ibitonyanga bya elution biri hagati ya membrane, kugirango biveho rwose ADN ihambiriye.

9. Nyuma yo kongeramo eluent, irashobora guterurwa mubwogero bwamazi bwa dogere 55 muminota 5 cyangwa ugashyirwa mubwogero bwamazi bwa dogere 50 muminota irenga 10, cyangwa ugafungirwa hamwe na parafilm kuri dogere 4 ijoro ryose, hanyuma ugashyiramo centrifug kugirango ukire bukeye, ingaruka nibyiza.

10. Ongeraho centrifuged eluate isubire kumurongo wa adsorption na centrifuge ongera.

Uburyo burambuye nuburyo bwo kugarura ibicuruzwa bya PCR

1. Rubber isanzwe

Niba ushaka kugarura kole, nibyiza gukoresha igikoresho, cyoroshye kandi gifite igipimo cyo hejuru cyo gukira.Niba ukeneye rwose kugarura intoki, urashobora kongeramo inshuro 3 ingano ya TE nyuma yo gukata kole.Nyuma yo gushonga mu bwogero bwamazi, fenol, fenol / chloroform ikuramo neza, hanyuma Ethanol ikagwa.Nibyo.

2. Kugarura ADN kuva geli yo gushonga

Kweza ibice bya ADN Ongeramo TE (10 mmol / l Tris-HCl pH8.0, 0.1 mmol / l EDTA) bingana nubunini bwa gel, hanyuma ushire mubwogero bwamazi 65 ° C muminota 5 kugirango ushonga burundu.

Nyuma yo kuzanwa mubushyuhe bwicyumba, ingano ingana na fenol (yuzuzwa na TE, TE yafunzwe mugice cyo hejuru, hanyuma igice cyo hepfo ya fenol ikurwaho) hiyongeraho, hanyuma imvange ivangwa gahoro gahoro (nta kuvanga bisabwa), hanyuma yinjizamo 12,000 rpm muminota 3.Subiramo inshuro 1-2.

Fata ndengakamere, ongeramo 0.1 ingano ya acetate ya sodium ya 3mol / L (pH 5.2) hamwe ninshuro 2,5 za etanol yuzuye kugirango ukore imvura ya Ethanol.Gabanya ADN yatunganijwe hamwe na TE ikwiye, upime ibirimo, kandi witegure gukoreshwa (irashobora gukoreshwa muburyo bwo gusesengura imiterere ya gene, gutegura iperereza, nibindi).

3. PCR isubirana hamwe na amplification nziza yihariye

Niba umwihariko wa amplification ya PCR ari nziza, nibyiza gusa byo kwezwa no kugarura ibicuruzwa bya PCR.Urashobora kongeramo 50ug / ml proteinase K kubicuruzwa bya PCR, dogere 37 kuri 1h, gukuramo rimwe hamwe na fenol / chloroform, gukuramo rimwe hamwe na chloroform, hanyuma ukongeramo 0.1 ingano yindengakamere.Sodium acetate yagaruwe nubushyuhe hamwe nubunini bwa 2,5 bya Ethanol yuzuye.

Ibicuruzwa bifitanye isano:

https://www.foreivd.com/pcr-purification-kit-2-umusaruro/

https://www.foreivd.com/amaraso-superdirecttm-pcr-kit-edta-product/