Iingcebiso zokuphucula ukuBuyiselwa kweGlue

1. Ukwandisa umthwalo wesampuli ngexesha le-electrophoresis.

2. Sebenzisa i-electrophoresis buffer esanda kulungiswa.

3. Xa usika i-glue, zama ukusika kuphela iglue ngeentambo zokunciphisa umthamo wokusika iglue: ungadingi iglue kunye neengcezu ezimbalwa zenjongo, ngaphandle koko kuya kuchaphazela izinga lokubuyisela.

4. Emva kokuncibilikisa iinqununu ezimbini okanye ngaphezulu zeglue, sebenzisa ityhubhu kungakhathaliseki ukuba ivolumu inkulu kangakanani kwaye uyithumele kwikholamu efanayo.

5. Isisombululo esongeziweyo kwi-sol sinokuba yinto encinci ngakumbi, ehambelana nokubopha i-DNA kwi-membrane, kodwa ngokuqhelekileyo ayidluli i-750ul.

6. Isitshixo sokubuyisela i-gel kukubopha i-DNA kwikholomu ngokusebenzisa i-salt concentration, i-acidity (intlawulo) kunye ne-hydrophobicity yesisombululo kwikholamu.Ngoko ke, ukuba i-pH ye-electrophoresis buffer iphezulu kakhulu, i-10ul (pH 5.0, 3mol / L NaAC) inokongezwa kwi-sol;ukuze uthintele ngcono iimolekyuli ze-DNA kwi-membrane, i-30% isopropanol inokongezwa kubushushu kulwelo emva kokunyibilikisa iglu.

7. Ngaphambi kokuba ungeze i-eluent, shiya ikholamu kwindawo yokushisa kwegumbi imizuzu embalwa (malunga nemizuzu eyi-10) ukucima ngokupheleleyo i-ethanol.

8. Ekugqibeleni, yongeza i-eluent encinci ukunciphisa umthamo wokubuyisela.Ngokubanzi, i-30-50μl eluent isetyenziselwa elution (hayi kancinci kakhulu, kungenjalo ayiyi kukwazi ukumanzisa inwebu, engabangeli lution);amathontsi elution kumbindi inwebu, ukuba elute ngokupheleleyo DNA ebotshelelwe inwebu.

9. Emva kokongeza i-eluent, inokuthi ikhutshwe kwindawo yokuhlambela amanzi e-55 degrees imizuzu emi-5 okanye ifakwe kwindawo yokuhlambela amanzi e-degrees ezingama-50 ngaphezu kwemizuzu eyi-10, okanye itywinwe ngeparafilm kwi-4 degrees ngobusuku, kwaye emva koko i-centrifuged yokubuyisela ngosuku olulandelayo, umphumo ulungile.

10.Yongeza i-centrifuged eluate emva kwikholamu ye-adsorption kunye ne-centrifuge kwakhona.

Iindlela ezineenkcukacha kunye neenkqubo zokubuyiselwa kwemveliso ye-PCR

1. Ukurisayikilishwa kwerabha eqhelekileyo

Ukuba ufuna ukubuyisela iglue, kungcono ukusebenzisa ikiti, efanelekileyo kwaye inezinga eliphezulu lokubuyisela.Ukuba ufuna ngokwenene ukuyibuyisela ngesandla, unokongeza amaxesha ama-3 umthamo we-TE emva kokusika iglue.Emva kokunyibilika kwindawo yokuhlambela amanzi, i-phenol, i-phenol / i-chloroform ikhutshwe ngokucocekileyo, kwaye i-ethanol iyancipha.Yiyo leyo.

2. Ukubuyiselwa kwe-DNA kwi-gel ephantsi yokunyibilika

Ukuhlanjululwa kwe-DNA Fragments Yongeza i-TE (10 mmol / l Tris-HCl pH8.0, 0.1 mmol / l EDTA) ilingana nomthamo wejel, kwaye ubeke kwindawo yokuhlambela amanzi angama-65 ° C imizuzu emi-5 ukutshabalalisa i-gel ngokupheleleyo.

Emva kokuba iziswe kwiqondo lokushisa, inani elilinganayo le-phenol (igcwele i-TE, i-TE ivalwe kwinqanaba eliphezulu, kwaye i-layer ephantsi ye-phenol isuswe) yongezwa, kwaye umxube wawuxutywe ngobumnene (akukho mxube ofunekayo), kunye ne-centrifuged kwi-12,000 rpm imizuzu ye-3.Phinda 1-2 amaxesha.

Thatha i-supernatant, yongeza i-0.1 ivolumu ye-3mol / L ye-acetate ye-sodium (pH 5.2) kunye namaxesha e-2.5 umthamo we-ethanol ngokupheleleyo ukwenza imvula ye-ethanol.Ukunyibilikisa i-DNA ecocekileyo kunye nenani elifanelekileyo le-TE, ukulinganisa umxholo, kwaye ulungiselele ukusetyenziswa (ingasetyenziselwa uhlalutyo lwesakhiwo se-gene, ukulungiswa kweprobe, njl.).

3. Ukubuyiselwa kwe-PCR kunye neenkcukacha ezilungileyo zokukhulisa

Ukuba i-speciality ye-PCR yokukhulisa ilungile, yinto nje yokucocwa kunye nokubuyiswa kwemveliso ye-PCR.Unokongeza i-50ug / ml proteinase K kwimveliso ye-PCR, i-37 degrees ye-1h, ikhuphe kanye nge-phenol / chloroform, ikhuphe kanye nge-chloroform, kwaye ungeze i-0.1 ivolumu ye-supernatant.I-acetate ye-sodium yafunyanwa yimvula kunye ne-2.5 imiqulu ye-ethanol epheleleyo.

Iimveliso eziyeleleneyo:

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

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