DNA Extraction Mini Kit

Iimeko zokugcina

Gcina kwi -15 ~ -25 ℃ kunye nokuthutha kwiqondo lokushisa.

Amacandelo

Buffer GDP:I-DNA ebophelelayo isithinteli.

Isithinteli GW:Isithinteli sokuhlamba;yongeza i-ethanol epheleleyo ngevolumu ebonisiweyo kwibhotile ngaphambi kokusetyenziswa.

Elution Buffer:Uvavanyo.

I-FastPure DNA Mini Columns-G:Iintsika ze-adsorption ze-DNA.

Imibhobho yoQokelelo 2 ml:Iityhubhu zokuqokelelwa kokucoca.

Izinto Ezilungisiweyo

I-1.5 ml iityhubhu ze-sterilized, i-ethanol epheleleyo kunye ne-isopropanol (xa i-DNA fragment ≤100 bp, yongeza ivolumu ye-1

I-isopropanol ukuya kwi-gel yomthamo we-1), ukuhlamba amanzi.

Inkqubo yoVavanyo

Yongeza i-80 ml ye-ethanol ukuhlambulula i-Buffer GW njengoko kubonisiwe kwithegi ngaphambi kokusetyenziswa, gcina kwiqondo lokushisa.

Inkqubo

1. PCR reaction solution

Iskimu sokutsalwa kwegel:Yongeza umthamo olinganayo weBuffer GDP PCR isikimu sokubuyisela isisombululo:Yongeza ka-5 isithinteli sevolyum

2. I-GDP Bala umthamo wejeli (100  μNdilingana no 100 mg )

Dibanisa ijeli

3. Tshisa nge-50 ~ 55℃

4. Bopha Hlamba

Yongeza i-300 μL ye-Buffer GDP*

Yongeza i-700 μL ye-Buffer GW

Yongeza i-700 μL ye-Buffer GW

5. Elute

Yongeza i-20 – 30μL ye-Elution Buffer okanye amanzi adiyoni

Amanqaku* PCR reaction recovery fluid ngaphandle kweli nyathelo

Inkqubo yokukhutshwa kwegel

1. Emva kwe-DNA electrophoresis yokwahlulahlula amaqhekeza e-DNA, cima umgca omnye weqhekeza le-DNA kwijeli ye-agarose phantsi kokukhanya kwe-UV.Kucetyiswa ukuba usebenzise iphepha elifunxayo ukufunxa ukufuma okubonakalayo kwejeli kwaye unciphise ubungakanani besilayi sejeli ngokususa iagarose eyongezelelweyo kangangoko unako.Ukulinganisa i-gel slice (ngaphandle ityhubhu ye-microcentrifuge) ukubala umthamo wayo: Umthamo we-gelslice ye-100 mg malunga ne-100 μL, ucinga ukuba ubuninzi be-1g / ml.

2. Yongeza umthamo olinganayo we-Buffer GDP, incubate kwi-50 ~ 55 ℃ kwi-7-10 min (ngokobukhulu be-gel, lungisa ixesha lokufakwa kwe-incubation de i-gel ichithwe ngokupheleleyo).Guqula ityhubhu ngamaxesha e-2 ngexesha lokufukamela.

Δ Ukongezwa kwemiqulu ye-1-3 ye-Buffer GDP akuyi kuba nefuthe kwi-DNA yokubuyisela ukusebenza kakuhle.Ukuba iqhekeza le-DNA liza kufunyanwa <100 bp, imiqulu ye-3 ye-Buffer GDP kufuneka yongezwe;xa i-gel slice ichithwe ngokupheleleyo, yongeza i-1 ivolumu ye-isopropanol kwaye udibanise ngokucokisekileyo, uze uqhubeke kwisinyathelo esilandelayo.

3. I-Centrifuge ngokufutshane ukuzisa isampuli ukuya ezantsi kumbhobho, faka i-FastPure DNA Mini Columns-G kwiiThubhu zokuQokelelwa kwe-2 ml, udlulisele ngononophelo isisombululo esiphezulu se-700 μL kanye

ixesha kwiikholamu zokucoca, i-centrifuge kwi-12,000 rpm (13,800 X g) ye-30-60 sec.

4. Lahla i-filtrate kwaye ungeze i-300 μL ye-Buffer GDP kwikholomu, faka kwiqondo lokushisa kwi-1 min, i-centrifuge kwi-12,000 rpm (13,800 X g) ye-30-60 sec.

5. Lahla i-filtrate kwaye wongeze i-700 μL ye-Buffer GW (jonga ukuba i-ethanol epheleleyo yongezwe kwangaphambili!) kwikholamu, i-centrifuge kwi-12,000 rpm (13,800 X g) kwi-30-60 sec.

Δ Nceda wongeze i-Buffer GW malunga nodonga lwekholamu ye-adsorption, okanye wongeze i-Buffer GW isiciko sangasemva kwaye usixube sijonge phantsi kangange-2 - 3 amaxesha ukunceda ukugungxula ngokupheleleyo ityuwa encamathele kudonga lwetyhubhu.

6. Phinda inyathelo lesi-5.

Δ Ukugungxulwa nge-Buffer GW kabini kunokuqinisekisa ukuba ityuwa isuswe ngokupheleleyo kwaye isuse impembelelo kwimifuniselo elandelayo.

7. Lahla i-filtrate kunye ne-centrifuge ikholamu engenanto kwi-12,000 rpm (13,800 X g) kwi-2 min.

8. Faka ikholamu kwi-tube ecocekileyo ye-1.5 ml ye-microcentrifuge, yongeza i-20 - 30 μL ye-Elution Buffer ukuya kumbindi we-membrane yekholomu, incubate i-2 min, kwaye emva koko i-centrifuge kwi-12,000 rpm (13,800 X g) kwi-1 min.Lahla ikholamu, gcina iDNA efunyenweyo kwi -20.

Δ Ukutshintshela i-supernatant yenyathelo lesi-8 kwikholamu ukuze iphinde iphinde ifudumale i-Elution Buffer ukuya kwi-55 (xa iqhekeza le-DNA> 3 kb) inokuba luncedo ukunyusa ukusebenza kakuhle kokubuyisela.

Inkqubo yokubuyisela iimveliso zePCR

Lo mthetho usetyenziswa ekucoceni amaqhekeza e-DNA kwiimveliso ze-PCR, inkqubo ye-enzymatic reaction kunye nezinye iimveliso ezikrwada ze-DNA (kubandakanywa ne-DNA yofuzo).Esi sisombululo sinokususa ngokufanelekileyo i-nucleotides eyahlukeneyo, i-primers, i-primer dimers, i-molecule yetyuwa, i-enzymes kunye nezinye izinto ezingcolileyo.

1. Ngokufutshane iimveliso ze-PCR ze-centrifuge, isisombululo se-enzymatic reaction, kunye nezinye iimveliso ezikrwada ze-DNA.Qikelela umthamo wabo ngepipette kwaye udlulisele kwi-sterilized 1.5 ml okanye 2 ml ityhubhu.Yongeza i-ddH2O de ivolyum ifike kwi-100 μL;ngelixa i-DNA ye-genomic egxininise kakhulu, ukuhlanjululwa kwi-300 μL nge-ddH2O kuya kunceda ukuphucula ukusebenza kakuhle.

2. Yongeza imiqulu ye-5 ye-Buffer GDP, xuba ngokucokisekileyo ngokuguqula okanye ukuguqula.Ukuba i-DNA fragment of interest>100 bp, ezongezelelweyo 1.5 volumes (isampulu + Buffer GDP) ye-ethanol kufuneka yongezwe.

3. Faka kwakhona ikholamu kwi-tube yokuqokelela, udlulisele i-mixtrue kwikholomu, i-centrifuge kwi-12,000 rpm (13,800 × g) ye-30 - 60 sec.Ukuba umthamo wesisombululo esixutyiweyo ngu> 700 µL, buyisela ikholamu ye-adsorption kwityhubhu yokuqokelela, udlulisele isisombululo esiseleyo kwikholamu ye-adsorption, kunye ne-centrifuge ku-12,000 rpm (13,800 × g) kwi-30 - 60 sec.

4. Intsebenzo elandelayo ibhekisa kwinyathelo lesi-5 – lesi-8 le-08- 1/iprogram yokutsalwa kwegel.