Mazano eKuvandudza Glue Recovery

1. Wedzera muenzaniso wekutakura panguva ye electrophoresis.

2. Shandisa ichangobva kugadzirwa electrophoresis buffer.

3. Paunenge uchicheka glue, edza kucheka glue chete nemashizha kuti uderedze huwandu hwekucheka glue: usade glue nezvikamu zvishoma zvechinangwa, kana zvisina kudaro zvinokanganisa chiyero chekudzorera.

4. Mushure mekunyungudusa zvidimbu zviviri kana kupfuura zveglue, shandisa chubhu pasinei nokuti yakakura sei inzwi uye uiendese kune imwe chete.

5. Mushonga wakawedzerwa mu sol unogona kuva zvishoma, izvo zvinowedzera kusungirirwa kweDNA kune membrane, asi kazhinji haipfuuri 750ul.

6. Chinokosha chekugadzirisa gel ndechekusunga DNA kumutsara kuburikidza nehuwandu hwemunyu, acidity (charge) uye hydrophobicity yemhinduro mumutsara.Nokudaro, kana pH ye electrophoresis buffer yakanyanya, 10ul (pH 5.0, 3mol / L NaAC) inogona kuwedzerwa kune sol;kuitira kuti utore zvirinani mamorekuru eDNA pa membrane, 30% isopropanol inogona kuwedzerwa kupisa mukati memvura mushure mekunyungudutsa glue.

7. Usati wawedzera mutsara, siya mbiru panzvimbo yekushisa kwemaminetsi mashomanana (anenge maminetsi gumi) kuti ibudise zvakakwana ethanol.

8. Pakupedzisira, wedzera zvishoma zvishoma kuti uderedze huwandu hwekudzoreredza.Kazhinji, 30-50μl eluent inoshandiswa pakujekesa (kwete zvishoma, kana zvisizvo haizokwanisi kunyorovesa membrane, iyo isingagadzirisi kujekesa);madonhwe eelution ari pakati pe membrane, kuti abudise zvizere DNA yakasungwa kune membrane.

9. Mushure mekuwedzera kuvharwa, inogona kubudiswa mumvura yekushambidza ye55 maminetsi e55 kana kuiswa mumvura yekugeza ye50 degrees kwemaminitsi anopfuura gumi, kana kuvharwa neparafilm pa4 degrees usiku hwose, uye ipapo centrifuged kuti apore zuva rinotevera, mhedzisiro yacho yakanaka.

10.Wedzera iyo centrifuged eluate kumashure kune adsorption column uye centrifuge zvakare.

Yakadzama nzira uye maitiro ePCR chigadzirwa kudzoreredza

1. Zvakajairika rabha recycling

Kana iwe uchida kudzoreredza glue, zviri nani kushandisa kititi, iyo iri nyore uye ine yakakwira zvishoma kudzoreredza mwero.Kana iwe uchinyatsoda kuidzosera nemaoko, unogona kuwedzera katatu vhoriyamu yeTE mushure mekucheka glue.Mushure mekunyunguduka mumvura yekugezera, phenol, phenol / chloroform inotorwa zvakachena, uye ethanol inonaya.Ndizvo.

2. DNA kupora kubva pasi yakaderera melting point gels

Kucheneswa kweDNA Zvimedu Wedzera TE (10 mmol / l Tris-HCl pH8.0, 0.1 mmol / l EDTA) yakaenzana nehuwandu hwejeri, uye uise mubhati yemvura ye65 ° C kwemaminitsi mashanu kuti uparadze gel zvachose.

Mushure mokunge yaunzwa kukushisa kwekamuri, chiyero chakaenzana che phenol (yakazara neTE, TE yakavharwa mumucheka wepamusoro, uye chikamu chepasi che phenol chakabviswa) chakawedzerwa, uye musanganiswa wacho wakanyatsosanganiswa (pasina kusanganiswa kunodiwa), uye centrifuged pa 12,000 rpm kwemaminitsi matatu.Dzokorora 1-2 nguva.

Tora supernatant, wedzera 0.1 vhoriyamu ye3mol/L sodium acetate (pH 5.2) uye 2.5 times volume yeabsolute ethanol kuita ethanol kunaya.Bvisa DNA yakacheneswa nehuwandu hwakakodzera hweTE, kuyera zviri mukati, uye gadzirira kushandiswa (inogona kushandiswa pachinangwa chekuongorora maitiro emajini, kugadzirira probe, nezvimwewo).

3. PCR kupora ine yakanaka amplification specificity

Kana iyo chaiyo yePCR amplification yakanaka, ingori nyore kucheneswa uye kudzoreredza kwePCR chigadzirwa.Iwe unogona kuwedzera 50ug/ml proteinase K kuPCR chigadzirwa, 37 madhigirii kwe1h, kubvisa kamwe chete ne phenol/chloroform, kubvisa kamwe chete nekloroform, uye kuwedzera 0.1 vhoriyamu yemushura.Iyo sodium acetate yakadzoserwa nekunaya kwemvura ine 2.5 mavhoriyamu e absolute ethanol.

Zvigadzirwa zvinoenderana:

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

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