Ndụmọdụ maka ịkwalite mgbake gluu

1. Na-abawanye ibu sample n'oge electrophoresis.

2. Jiri ihe nchekwa electrophoresis akwadoro ọhụrụ.

3. Mgbe ị na-egbutu gluu, gbalịa ịkpụ naanị gluu na ibe iji belata olu nke ịcha ihe: adịghị mkpa gluu na iberibe nzube ole na ole, ma ọ bụghị ya, ọ ga-emetụta ọnụego mgbake.

4. Mgbe agbazechara abụọ ma ọ bụ karịa nke gluu, jiri tube n'agbanyeghị oke olu ma nyefee ya na otu kọlụm.

5. Ngwọta agbakwunyere na sol nwere ike ịbụ ntakịrị ihe, nke na-enye aka na njikọ DNA na akpụkpọ ahụ, ma n'ozuzu adịghị agafe 750ul.

6. Isi ihe maka mgbake gel bụ ijikọ DNA na kọlụm site na ntinye nnu, acidity (ụgwọ) na hydrophobicity nke ngwọta na kọlụm.Ya mere, ọ bụrụ na pH nke electrophoresis buffer dị oke elu, 10ul (pH 5.0, 3mol / L NaAC) nwere ike ịgbakwunye na sol;iji mee ka mkpụrụ ndụ DNA dịkwuo mma na akpụkpọ ahụ, 30% isopropanol nwere ike ịgbakwunye ọkụ n'ime mmiri mmiri mgbe ọ gbasasịrị gluu.

7. Tupu ịtinye eluent, hapụ kọlụm na okpomọkụ ụlọ maka nkeji ole na ole (ihe dị ka nkeji 10) iji kpochapụ ethanol kpamkpam.

8. N'ikpeazụ, tinye obere eluent iji belata olu mgbake.N'ozuzu, a na-eji 30-50μl eluent eme ihe maka elution (ọ bụghị obere, ma ọ bụghị ya, ọ gaghị enwe ike ime ka akpụkpọ ahụ dị nro, nke na-adịghị mma maka elution);ụmụ irighiri mmiri nke elution dị n'etiti akpụkpọ ahụ, iji kpochapụ DNA zuru oke na akpụkpọ ahụ.

9. Mgbe agbakwunyere eluent ahụ, enwere ike ịpụ ya na mmiri ịsa mmiri nke 55 degrees maka nkeji 5 ma ọ bụ tinye ya na mmiri ịsa mmiri nke 50 degrees maka ihe karịrị nkeji 10, ma ọ bụ mechie ya na parafilm na 4 degrees n'otu ntabi anya, wee centrifuged maka mgbake n'echi ya, mmetụta dị mma.

10. Tinye centrifuged eluate azụ na kọlụm mgbasa ozi na centrifuge ọzọ.

Ụzọ na usoro zuru ezu maka PCR ngwaahịa mgbake

1. Nrụgharị roba nkịtị

Ọ bụrụ na ịchọrọ ịgbake gluu, ọ kacha mma iji ngwa ngwa, nke dị mma ma nwee ntakịrị mgbake dị elu.Ọ bụrụ n'ezie na ịchọrọ iji aka gị nwetaghachi ya, ịnwere ike ịgbakwunye 3 ugboro olu TE mgbe ịchacha gluu.Mgbe agbazechara na mmiri ịsa ahụ, phenol, phenol/chloroform na-amịpụta nke ọma, ethanol na-ebulikwa ya.Ọ bụ ya.

2. DNA mgbake site na gels dị ala na-agbaze

Nchacha nke iberibe DNA Tinye TE (10 mmol/l Tris-HCl pH8.0, 0.1 mmol/l EDTA) hà nhata nke gel, ma tinye ya na mmiri mmiri 65 Celsius C maka nkeji 5 ka ọ gbazee gel kpamkpam.

Mgbe e webatara ya n'ime ụlọ okpomọkụ, nha nha nha nke phenol (jupụtara na TE, TE na-emechi na oyi akwa nke elu, na-ewepụkwa obere oyi akwa nke phenol), ma jiri nwayọọ gwakọta ngwakọta ahụ (ọ dịghị ngwakọta achọrọ), na centrifuged na 12,000 rpm maka nkeji 3.Tinyegharịa ugboro 1-2.

Were supernatant, tinye 0.1 olu nke 3mol/L sodium acetate (pH 5.2) na 2.5 ugboro olu nke zuru oke ethanol iji mepụta ethanol ọdịda.Jiri TE kwesịrị ekwesị gbaze DNA ahụ emechara, tụọ ọdịnaya ya ma kwadoo maka ojiji (enwere ike iji ya maka nyocha nhazi usoro mkpụrụ ndụ ihe, nkwadebe nyocha, wdg).

3. PCR mgbake na ezigbo nkwalite nkọwa

Ọ bụrụ na nkọwapụta nke nkwalite PCR dị mma, ọ bụ naanị nchacha na mgbake nke ngwaahịa PCR.Ị nwere ike tinye 50ug/ml proteinase K na ngwaahịa PCR, 37 degrees maka 1h, wepụ otu ugboro na phenol/chloroform, wepụ otu ugboro na chloroform, tinye 0.1 olu nke supernatant.Enwetara sodium acetate site na mmiri ozuzo na mpịakọta 2.5 nke ethanol zuru oke.

Ngwaahịa ndị emetụtara:

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

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