Mini Kit ɗin hakar DNA
Wannan kit ɗin yana ɗaukar ingantacciyar tsarin buffer da fasahar tsarkakewa na silica gel, wanda zai iya dawo da gutsuttsuran DNA 70 bp - 20 kb daga tarin tarin TAE ko TBE agarose gel.Rukunin adsorption na DNA na iya tallata DNA musamman a ƙarƙashin yanayin gishiri mai girma.Bugu da kari, kit ɗin na iya tsarkake gutsuttsuran DNA kai tsaye daga samfuran PCR, tsarin amsawar enzymatic ko samfuran ɗanyen DNA da aka samu ta wasu hanyoyin, da kuma cire ƙazanta irin su sunadaran, sauran mahadi, ions gishiri na inorganic da oligonucleotide primers.Yana iya tabbatar da cewa za a iya kammala tsarkakewa a cikin 10-15min.Ana iya amfani da DNA ɗin da aka tsarkake kai tsaye don ligation, canzawa, narkewar enzyme, fassarar in vitro, PCR, sequencing, microinjection, da sauransu.
Yanayin ajiya
Adana a -15 ~ -25 ℃ da jigilar kaya a dakin da zafin jiki.
Abubuwan da aka gyara
| Abubuwan da aka gyara | (100 rxns) |
| Buffer GDP | ml 80 |
| Buffer GW | 2 × 20 ml |
| Elution Buffer | 20 ml |
| FastPure DNA Mini Columns-G | 100 |
Babban GDP:DNA daure buffer.
Buffer GW:Wurin wankewa;ƙara cikakken ethanol ta ƙarar da aka nuna akan kwalban kafin amfani.
Matsakaicin Haske:Haske.
FastPure DNA Mini Columns-G:ginshiƙan tallan DNA.
Tarin Tubus 2 ml:Tarin bututu don tacewa.
Shirye-shiryen Kayayyakin
1.5 ml haifuwa bututu, cikakken ethanol da isopropanol (lokacin da DNA gutsuttsura ≤100 bp, ƙara 1 girma
isopropanol zuwa gel 1 girma), wanka na ruwa.
Tsarin Gwaji
Ƙara 80 ml na ethanol don tsarma Buffer GW kamar yadda aka nuna akan tag kafin amfani, adana a zafin jiki.
Makanikai
1. Maganin amsawar PCR
Tsarin hakar Gel: Ƙara daidaitaccen girma mai buffer GDP PCR tsarin dawo da martani:Ƙara sau 5 ƙarar Matsala
2. GDP Ƙididdigar girman gel (100 μl daidai 100 MG)
Narke gel
3. Yi zafi a 50 ~ 55℃
4. Daure Wanke
Ƙara 300 μL na GDP na Buffer*
Ƙara 700 μL na Buffer GW
Ƙara 700 μL na Buffer GW
5. Elute
Ƙara 20 - 30μL na Elution Buffer ko ruwan da aka cire
Bayanan kula * PCR dauki dawo da ruwa ba tare da wannan matakin ba
Shirin hakar gel
1. Bayan DNA electrophoresis don ɓarna gutsuttsuran DNA, fitar da ɗigon DNA guda ɗaya daga gel na agarose a ƙarƙashin hasken UV.Ana ba da shawarar yin amfani da takarda mai shayarwa don shayar da danshin gel na fili kuma a rage girman yanki ta hanyar cire ƙarin agarose kamar yadda za ku iya.Yi la'akari da yanki na gel (ba tare da bututun microcentrifuge ba) don ƙididdige ƙarar sa: Girman 100 MG gelslice yana da kusan 100 μL, ɗauka cewa yawancin shine 1g/ml.
2. Ƙara daidai girman buffer GDP, incubate a 50 ~ 55 ℃ don 7-10 min (bisa ga girman gel, daidaita lokacin shiryawa har sai gel gaba ɗaya ya narkar da).Juya bututu sau 2 yayin shiryawa.
∆ Ƙarin juzu'i na 1-3 na Buffer GDP ba zai tasiri ingancin dawo da DNA ba.Idan guntun DNA da za a dawo da shi <100 bp, ana buƙatar ƙara 3 juzu'i na Buffer GDP;lokacin da yanki na gel ya narkar da gaba ɗaya, ƙara ƙarar 1 na isopropanol da haɗuwa sosai, sannan ci gaba zuwa mataki na gaba.
3. Centrifuge a taƙaice don kawo samfurin zuwa kasan bututu, saka FastPure DNA Mini Columns-G a cikin Tarin Tubes 2 ml, a hankali canja wurin maganin a maximally na 700 μL sau ɗaya a.
lokaci zuwa ginshiƙan tacewa, centrifuge a 12,000 rpm (13,800 X g) don 30-60 sec.
4. Yi watsi da tacewa kuma ƙara 300 μL na Buffer GDP zuwa ginshiƙi, sanyawa a dakin da zafin jiki na 1 min, centrifuge a 12,000 rpm (13,800 X g) don 30-60 sec.
5. Yi watsi da tacewa kuma ƙara 700 μL na Buffer GW (duba idan an ƙara cikakken ethanol a gaba!) zuwa shafi, centrifuge a 12,000 rpm (13,800 X g) don 30-60 sec.
∆ Da fatan za a ƙara GW mai buffer a kusa da bangon ginshiƙi na talla, ko ƙara murfin baya na GW mai buffer a haɗa shi sama da sau 2 – 3 don taimakawa gabaɗaya zubar gishirin da ke manne da bangon bututu.
6. Maimaita mataki na 5.
Δ Flushing tare da Buffer GW sau biyu zai iya tabbatar da cewa an cire gishiri gaba daya kuma ya kawar da tasiri akan gwaje-gwaje na gaba.
7. Yi watsi da tacewa kuma saka ginshiƙan fanko a 12,000 rpm (13,800 X g) don 2 min.
8. Saka ginshiƙi a cikin bututun microcentrifuge mai tsabta na 1.5 ml, ƙara 20 - 30 μL na Elution Buffer zuwa tsakiyar membrane na ginshiƙi, sanya shi don 2 min, sannan centrifuge a 12,000 rpm (13,800 X g) don 1 min.Yi watsi da ginshiƙi, adana DNA da aka samu a -20.
∆ Canja wurin babban mataki na 8 zuwa ginshiƙi don sake haɓakawa da preheating da Elution Buffer zuwa 55 (lokacin da guntun DNA> 3 kb) na iya taimakawa don haɓaka haɓakar farfadowa.
PCR kayayyakin dawo da shirin
Wannan ƙa'idar tana aiki don tsarkake gutsuttsura DNA daga samfuran PCR, tsarin amsawar enzymatic da sauran samfuran ɗanyen DNA (ciki har da DNA na kwayoyin halitta).Wannan bayani zai iya dacewar cire nau'ikan nucleotides, abubuwan farko, dimers na farko, kwayoyin gishiri, enzymes da sauran datti.
1. A taƙaice centrifuge PCR kayayyakin, enzymatic dauki bayani, da sauran DNA danyen kayayyakin.Yi ƙididdige ƙarar su tare da pipette kuma canja wurin zuwa bututun 1.5 ml ko 2 ml da aka haifuwa.Ƙara ddH2O har sai ƙarar har zuwa 100 μL;yayin da DNA na genomic tare da babban maida hankali, diluting zuwa 300 μL tare da ddH2O zai taimaka wajen inganta ingantaccen farfadowa.
2. Ƙara juzu'i 5 na Buffer GDP, gauraya sosai ta hanyar jujjuyawa ko jujjuyawa.Idan guntun DNA na sha'awa> 100 bp, ana buƙatar ƙara ƙarin juzu'i 1.5 (samfurori + Buffer GDP) na ethanol.
3. Saka ginshiƙi baya cikin bututun tarin, canja wurin mixtrue zuwa shafi, centrifuge a 12,000 rpm (13,800 ×g) don 30 - 60 sec.Idan ƙarar bayani mai gauraya> 700 µL, sanya ginshiƙin tallan baya a cikin bututun tarin, canja wurin sauran bayani zuwa ginshiƙin talla, da centrifuge a 12,000 rpm (13,800 × g) don 30 - 60 sec.
4. Ayyukan na gaba yana nufin mataki na 5 - 8 na 08- 1 / shirin cire gel.
Aikace-aikace
Matsaloli daban-daban na TAE ko TBE agarose gel;Samfuran PCR, tsarin amsawar enzymatic ko wasu samfuran DNA na ɗanyen da aka samu ta hanyoyi daban-daban.Abubuwan da aka dawo dasu sun fito daga70 bp -20 kb.
Bayanan kula
Don bincike kawai amfani.Ba don amfani a cikin hanyoyin bincike ba.
1. Ƙara 80 ml na ethanol don tsarma Buffer GW kamar yadda aka nuna akan tag kafin amfani, adana a dakin da zafin jiki.
2. Idan Buffer GDP yana da sauƙi don haɓakawa a lokacin ajiyar ƙananan zafin jiki, ana iya sanya shi a cikin zafin jiki na wani lokaci kafin amfani.Idan ya cancanta, ana iya preheated a cikin wanka na ruwa na 37 ℃ har sai an narkar da hazo gaba daya, sannan ana iya amfani da shi bayan haɗuwa.
3. Saita zafin wanka na ruwa zuwa 50 ~ 55 ℃ a gaba.
4. A cikin shirin cirewa na 08-1 / Gel mataki na 1, rage girman girman yanki na gel zai rage girman lokacin narkar da kuma ƙara yawan farfadowa (DNA mai layi yana da sauƙi don hydrolyze lokacin da aka ci gaba da fallasa a babban zafin jiki).Kada a bijirar da gel DNA zuwa UV na dogon lokaci, saboda hasken ultraviolet zai iya haifar da lalacewar DNA.
5. Narkar da gel a cikin shirin 08-1 / gel cirewa mataki na 2 gaba daya, in ba haka ba za a yi tasiri sosai game da aikin dawo da DNA.
6. Preheat Elution Buffer ko ddH2O zuwa 55 ℃ , wanda ke taimakawa wajen inganta haɓakar DNA.Ana ba da shawarar adana DNA a cikin mafi girman 2.5 mM Tris-HCl, pH 7.0 - 8.5.
