Malangizo Othandizira Kubwezeretsa Glue

1. Wonjezerani chitsanzo cha katundu pa electrophoresis.

2. Gwiritsani ntchito chotchinga chatsopano cha electrophoresis.

3. Mukamadula guluu, yesetsani kudula guluu ndi mizere kuti muchepetse kuchuluka kwa guluu: osafunikira guluu wokhala ndi zidutswa zochepa, apo ayi zingakhudze kuchira.

4. Mukasungunula zidutswa ziwiri kapena zingapo za guluu, gwiritsani ntchito chubu mosasamala kanthu kuti voliyumuyo ndi yaikulu bwanji ndikusamutsira ku gawo lomwelo.

5. Njira yothetsera vutoli ikhoza kukhala yowonjezera pang'ono, yomwe imakhala yabwino kwambiri kumangiriza DNA ku nembanemba, koma kawirikawiri musapitirire 750ul.

6. Chinsinsi cha kuchira kwa gel ndikumanga DNA kumtunda kupyolera mu mchere wambiri, acidity (charge) ndi hydrophobicity ya yankho mu gawoli.Choncho, ngati pH ya electrophoresis buffer ndi yokwera kwambiri, 10ul (pH 5.0, 3mol / L NaAC) ikhoza kuwonjezeredwa ku sol;kuti athe kusokoneza mamolekyu a DNA pa nembanemba, 30% isopropanol ikhoza kuwonjezeredwa kutenthedwa mumadzimadzi pambuyo posungunula guluu.

7. Musanaphatikizepo, siyani mzatiwo pa kutentha kwa firiji kwa mphindi zingapo (pafupifupi mphindi 10) kuti musungunuke mowa.

8. Pomaliza, onjezani zocheperako kuti muchepetse kuchuluka kwa kuchira.Nthawi zambiri, 30-50μl eluent imagwiritsidwa ntchito pakuwunikira (osati pang'ono, apo ayi sichitha kunyowetsa nembanembayo, yomwe siingathandize kutulutsa);madontho a elution ali pakati pa nembanemba, kuti atulutse DNA yomangidwa ku nembanemba.

9. Pambuyo powonjezera chojambulacho, chikhoza kuchotsedwa m'madzi osamba a madigiri 55 kwa mphindi 5 kapena kuikidwa mumadzi osambira a madigiri 50 kwa mphindi zoposa 10, kapena kusindikizidwa ndi parafilm pa madigiri 4 usiku, ndiyeno centrifuged kuti muchiritse tsiku lotsatira, zotsatira zake ndi zabwino.

10.Onjezani centrifuged eluate kubwerera ku adsorption column ndi centrifuge kachiwiri.

Tsatanetsatane wa njira ndi njira zobwezeretsera zinthu za PCR

1. Kubwezeretsanso mphira wamba

Ngati mukufuna kubwezeretsa guluu, ndi bwino kugwiritsa ntchito zida, zomwe zimakhala zosavuta komanso zotsika pang'ono.Ngati mukufunadi kuchibwezeretsa pamanja, mutha kuwonjezera katatu kuchuluka kwa TE mutatha kudula guluu.Akasungunuka m'madzi osamba, phenol, phenol / chloroform amachotsedwa bwino, ndipo ethanol imatulutsa mpweya.Ndichoncho.

2. Kuchira kwa DNA kuchokera ku ma gels otsika osungunuka

Kuyeretsedwa kwa Zidutswa za DNA Onjezerani TE (10 mmol / l Tris-HCl pH8.0, 0.1 mmol / l EDTA) yofanana ndi voliyumu ya gel osakaniza, ndipo ikani mumadzi osambira a 65 ° C kwa mphindi zisanu kuti musungunuke gel osakaniza.

Pambuyo pa kubweretsedwa kutentha, chiwerengero chofanana cha phenol (chodzaza ndi TE, TE chinasindikizidwa pamtunda wapamwamba, ndipo gawo la pansi la phenol linachotsedwa) linawonjezeredwa, ndipo kusakaniza kunali kosakanikirana (palibe kusakaniza kofunikira), ndi centrifuged pa 12,000 rpm kwa mphindi zitatu.Bwerezani 1-2 nthawi.

Tengani champhamvu kwambiri, onjezerani 0.1 voliyumu ya 3mol/L sodium acetate (pH 5.2) ndi 2.5 kuwirikiza kuchuluka kwa mowa wamphumphu kuti mugwire mpweya wa ethanol.Sungunulani DNA yoyeretsedwa ndi kuchuluka koyenera kwa TE, kuyeza zomwe zili, ndikukonzekera kuti mugwiritse ntchito (itha kugwiritsidwa ntchito posanthula kapangidwe ka jini, kukonzekera kafukufuku, ndi zina).

3. Kuchira kwa PCR ndi tsatanetsatane wabwino wokulitsa

Ngati tsatanetsatane wa kukulitsa kwa PCR ndikwabwino, ndikungoyeretsa kosavuta komanso kuchira kwa PCR.Mutha kuwonjezera 50ug/ml proteinase K ku PCR, madigiri 37 kwa 1h, kuchotsa kamodzi ndi phenol/chloroform, kuchotsa kamodzi ndi chloroform, ndikuwonjezera 0,1 voliyumu yamphamvu kwambiri.Sodium acetate inapezedwanso ndi mvula ndi ma voliyumu 2.5 a ethanol absolute.

Zogwirizana nazo:

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

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