Amathiphu Okuthuthukisa Ukutholwa Kweglue

1. Ukwandisa umthwalo wesampula ngesikhathi se-electrophoresis.

2. Sebenzisa i-electrophoresis buffer esanda kulungiswa.

3. Uma usika i-glue, zama ukusika kuphela i-glue ngemichilo ukuze unciphise ivolumu yokusika i-glue: ungadingi i-glue ngezingcezu ezimbalwa zenhloso, ngaphandle kwalokho izothinta izinga lokubuyisela.

4. Ngemva kokuncibilikisa izingcezu ezimbili noma ngaphezulu zeglue, sebenzisa ithubhu kungakhathaliseki ukuthi ivolumu inkulu kangakanani bese uyidlulisela kukholomu efanayo.

5. Isixazululo esingezwe ku-sol singase sibe ngaphezulu kancane, okuhambisana kakhulu nokubopha i-DNA kulwelwesi, kodwa ngokuvamile ungadluli ku-750ul.

6. Isihluthulelo sokubuyisela ijeli ukubopha i-DNA kukholamu ngokusebenzisa ukugxila kukasawoti, i-acidity (inkokhelo) kanye ne-hydrophobicity yesisombululo kukholamu.Ngakho-ke, uma i-pH ye-electrophoresis buffer iphezulu kakhulu, i-10ul (pH 5.0, 3mol / L NaAC) ingangezwa ku-sol;ukuze kunqandwe kangcono ama-molecule e-DNA kulwelwesi, i-isopropanol engu-30% ingangezwa ekushiseni oketshezini ngemva kokuncibilikisa ingcina.

7. Ngaphambi kokwengeza i-ethanol, shiya ikholomu ekamelweni lokushisa imizuzu embalwa (cishe imizuzu eyi-10) ukuze i-ethanol ihwamuke ngokuphelele.

8. Okokugcina, engeza okuncane kakhulu ukuze unciphise ivolumu yokutakula.Ngokuvamile, i-30-50μl i-eluent isetshenziselwa i-lution (hhayi kancane kakhulu, ngaphandle kwalokho ngeke ikwazi ukumanzisa ulwelwesi, olungahambisani nokukhishwa);amaconsi e-elution aphakathi nendawo yolwelwesi, ukuze akhiphe ngokugcwele i-DNA eboshelwe kulwelwesi.

9. Ngemva kokwengeza i-eluent, ingasuswa ekugezeni kwamanzi ama-degree angu-55 imizuzu engu-5 noma ifakwe emanzini okugeza angama-degree angu-50 imizuzu engaphezu kwe-10, noma ivalwe nge-parafilm ngama-degree angu-4 ngobusuku obubodwa, bese i-centrifuged ukuze ilulame ngosuku olulandelayo, umphumela muhle.

10.Yengeza i-centrifuged eluate emuva kukholomu ye-adsorption kanye ne-centrifuge futhi.

Izindlela ezinemininingwane nezinqubo zokutholwa komkhiqizo we-PCR

1. Ukugaywa kabusha kwenjoloba okuvamile

Uma ufuna ukubuyisela iglue, kungcono ukusebenzisa ikhithi, elula futhi enezinga eliphezulu lokutakula.Uma udinga ngempela ukuyibuyisela ngesandla, ungakwazi ukwengeza izikhathi ezingu-3 umthamo we-TE ngemva kokusika ingcina.Ngemva kokuncibilika emanzini okugeza, i-phenol, i-phenol/i-chloroform ikhishwa ngokuhlanzekile, futhi i-ethanol iyawa.Yilokho kuphela.

2. Ukubuyiselwa kwe-DNA kusuka kumajeli aphansi ancibilika

Ukuhlanzwa Kwezingcezu Ze-DNA Faka i-TE (10 mmol/l Tris-HCl pH8.0, 0.1 mmol/l EDTA) elingana nomthamo wejeli, bese ufaka kubhavu wamanzi ongu-65°C imizuzu emi-5 ukuze uhlakaze ijeli ngokuphelele.

Ngemuva kokuthi ilethwe ekamelweni lokushisa, inani elilinganayo le-phenol (eligcwele i-TE, i-TE livalwe ungqimba olungaphezulu, futhi ungqimba olungezansi lwe-phenol lususiwe) lwengezwa, futhi ingxube ixutshwe ngobumnene (akukho ukuxuba okudingekayo), futhi i-centrifuged ku-12,000 rpm imizuzu engu-3.Phinda izikhathi ezingu-1-2.

Thatha i-supernatant, engeza ivolumu engu-0.1 ye-3mol/L ye-sodium acetate (pH 5.2) kanye nevolumu ephindwe izikhathi ezingu-2.5 ye-ethanol ephelele ukuze wenze imvula ye-ethanol.Chaza i-DNA ehlanzekile ngenani elifanele le-TE, ulinganise okuqukethwe, futhi ulungiselele ukusetshenziswa (ingasetshenziselwa ukuhlaziywa kwesakhiwo sofuzo, ukulungiswa kophenyo, njll.).

3. Ukutholwa kwe-PCR ngokucaciswa okuhle kokukhulisa

Uma ukucaciswa kokukhulisa i-PCR kukuhle, kumane kuwukuhlanzwa okulula nokutholwa komkhiqizo we-PCR.Ungakwazi ukwengeza u-50ug/ml proteinase K emkhiqizweni we-PCR, ama-degree angu-37 ngehora elingu-1, ukhiphe kanye nge-phenol/chloroform, ukhiphe kanye nge-chloroform, bese wengeza ivolumu engu-0.1 ye-supernatant.I-acetate yesodium itholwe yimvula enemiqulu engu-2.5 ye-ethanol ephelele.

Imikhiqizo ehlobene:

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

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