DNA Extraction Mini Kit
Setsi sena se sebelisa mokhoa o ntlafalitsoeng oa "buffer system" le thekenoloji ea tlhoekiso ea silika ea silika, e ka fumanang likhechana tsa DNA tse 70 - 20 kb ho tsoa mefuteng e fapaneng ea gel ea TAE kapa TBE agarose.DNA adsorption column e ka bapa DNA ka ho khetheha tlas'a boemo bo nang le letsoai le phahameng.Ho phaella moo, kit e ka hloekisa ka ho toba likaroloana tsa DNA lihlahisoa tsa PCR, mekhoa ea ho itšoara ka enzymatic kapa lihlahisoa tsa DNA tse sa tsoakoang tse fumanoang ka mekhoa e meng, 'me li tlose litšila tse kang liprotheine, metsoako e meng ea lintho tse phelang, li-ion tsa letsoai tsa inorganic le oligonucleotide primers.E ka etsa bonnete ba hore tlhoekiso e ka phethoa nakong ea 10-15min.DNA e hloekisitsoeng e ka sebelisoa ka ho toba bakeng sa ligation, phetoho, tšilo ea enzyme, in vitro transcription, PCR, sequencing, microinjection, joalo-joalo.
Maemo a polokelo
Boloka ho -15 ~ -25 ℃ 'me u tsamaee ka mocheso oa kamore.
Likaroloana
| Likaroloana | (100 rxns) |
| Tšireletso ea GDP | 80 ml |
| Sekhahla sa GW | 2 × 20 di ml |
| Elution Buffer | 20 ml |
| FastPure DNA Mini Columns-G | 100 |
Buffer GDP:DNA e tlamang buffer.
Buffer GW:Ho hlatsoa buffer;eketsa ethanol e felletseng ka molumo o bontšitsoeng botlolong pele o sebelisoa.
Elution Buffer:Elution.
FastPure DNA Mini Columns-G:Litšiea tsa adsorption tsa DNA.
Li-tubes tsa pokello 2 ml:Li-tubes tsa pokello bakeng sa filtrate.
Lintho Tse Lokisitsoeng
1.5 ml sterilized tubes, absolute ethanol le isopropanol (ha DNA fragment ≤100 bp, eketsa molumo o le mong
isopropanol ho 1 bophahamo ba gel), ho hlapa metsi.
Mokhoa oa ho Leka
Kenya 80 ml ea ethanol ho hlapolla Buffer GW joalo ka ha ho bonts'itsoe tag pele o sebelisoa, boloka mocheso oa kamore.
Mokhoa
1. PCR reaction solution
Sekema sa ho ntša Gel: Eketsa sekhahla se lekanang sa Buffer GDP PCR reaction recovery scheme:Kenya makhetlo a 5 ho feta Buffer
2. GDP Bala bophahamo ba gel (100 μl e lekana le 100 mg)
Hlakola gel
3. Preheat ka likhato tse 50-55℃
4. Tlama Hlatsoa
Kenya 300 μL ea Buffer GDP*
Kenya 700 μL ea Buffer GW
Kenya 700 μL ea Buffer GW
5. Elute
Kenya 20 - 30μL ea Elution Buffer kapa metsi a ntšitsoeng
Lintlha * PCR reaction fluid recovery ntle le mohato ona
Lenaneo la ho ntša li-gel
1. Ka mor'a DNA electrophoresis bakeng sa ho arola likaroloana tsa DNA, tlosa mola o le mong oa sekhechana sa DNA ho tloha gel ea agarose tlas'a lebone la UV.Ho khothalletsoa ho sebelisa pampiri e monyang ho monya mongobo o hlakileng oa gel le ho fokotsa boholo ba selae sa gel ka ho tlosa agarose e eketsehileng ka hohle kamoo u ka khonang.Beha selae sa gel (ntle le microcentrifuge tube) ho bala bophahamo ba eona: Bophahamo ba 100 mg gelslice ke hoo e ka bang 100 μL, ho nka hore boima ke 1g/ml.
2. Eketsa palo e lekanang ea Buffer GDP, e kenye ka 50 ~ 55 ℃ bakeng sa 7-10 min (ho ea ka boholo ba gel, fetola nako ea ho qhoqhoa ho fihlela gel e qhibiliha ka ho feletseng).Fetola tube ka makhetlo a 2 nakong ea ho kenya.
Δ Keketso ea li-volumes tse 1-3 tsa Buffer GDP e ke ke ea susumetsa katleho ea DNA ea ho hlaphoheloa.Haeba sekhechana sa DNA se tla khutlisoa <100 bp, meqolo e 3 ea Buffer GDP e hloka ho kenyelletsoa;ha selae sa gel se qhibilihile ka ho feletseng, eketsa 1 molumo oa isopropanol 'me u kopanye ka botlalo, ebe u tsoela pele mohato o latelang.
3. Centrifuge ka bokhutšoane ho tlisa sampuli ho ea tlase ea tube, kenya FastPure DNA Mini Columns-G ka har'a Li-tubes tsa Pokello ea 2 ml, fetisetsa tharollo ka hloko ka boholo ba 700 μL hang a
nako ea ho litšiea tsa filtration, centrifuge ho 12,000 rpm (13,800 X g) bakeng sa 30-60 sec.
4. Lahla filtrate 'me u kenye 300 μL ea Buffer GDP ho kholomo, e kene ka mocheso oa kamore bakeng sa 1 min, centrifuge ka 12,000 rpm (13,800 X g) bakeng sa 30-60 sec.
5. Lahla filtrate 'me u kenye 700 μL ea Buffer GW (hlahloba hore na ethanol e feletseng e kentsoe esale pele!) ho kholomo, centrifuge ka 12,000 rpm (13,800 X g) bakeng sa 30-60 sec.
Δ Ka kopo eketsa Buffer GW ho potoloha lebota la kholomo ea adsorption, kapa u kenye sekoaelo sa morao sa Buffer GW ebe u se kopanya ka holimo bakeng sa 2 - 3 linako tse ling ho thusa ho hlakola letsoai ka botlalo leboteng la tube.
6. Pheta mohato oa 5.
Δ Ho hlatsoa ka Buffer GW habeli ho ka netefatsa hore letsoai le tlosoa ka ho feletseng le ho felisa tšusumetso litekong tse latelang.
7. Lahla filtrate le centrifuge kholomo e se nang letho ho 12,000 rpm (13,800 X g) bakeng sa 2 min.
8. Kenya kholomo ka har'a tube e hloekileng ea 1.5 ml ea microcentrifuge, eketsa 20 - 30 μL ea Elution Buffer ho ea bohareng ba lera la likholomo, e kenye metsotso e 2, ebe centrifuge ka 12,000 rpm (13,800 X g) bakeng sa 1 min.Lahla kholomo, boloka DNA e fumanoeng ho -20 .
Δ Ho fetisetsa matla a holimo a mohato oa 8 ho kholumo hore e hlake hape le ho futhumatsa Elution Buffer ho 55 (ha sekhechana sa DNA> 3 kb) ho ka thusa ho eketsa ts'ebetso ea ho hlaphoheloa.
Lenaneo la ho hlaphoheloa ha lihlahisoa tsa PCR
Protocol ena e sebetsa ho hloekisa likhechana tsa DNA lihlahisoa tsa PCR, sistimi ea enzymatic reaction le lihlahisoa tse ling tsa DNA (ho kenyeletsoa le DNA ea lefutso).Tharollo ena e ka khona ho tlosa li-nucleotide tse fapaneng, li-primers, primer dimers, limolek'hule tsa letsoai, li-enzyme le litšila tse ling.
1. Ka bokhutšoanyane lihlahisoa tsa PCR tsa centrifuge, tharollo ea enzymatic reaction, le lihlahisoa tse ling tsa DNA crude.Hakanya bophahamo ba tsona ka pipette 'me u fetisetse ho sterilized 1.5 ml kapa 2 ml tube.Kenya ddH2O ho fihlela molumo o fihla ho 100 μL;athe genomic DNA e nang le mohopolo o phahameng, ho hlapolla ho 300 μL ka ddH2O ho tla thusa ho ntlafatsa ts'ebetso ea ho hlaphoheloa.
2. Eketsa li-volumes tse 5 tsa Buffer GDP, kopanya hantle ka ho inverting kapa vortexing.Haeba karolo ea DNA ea phaello> 100 bp, li-volumes tse ling tse 1.5 (lisampole + Buffer GDP) tsa ethanol li hloka ho eketsoa.
3. Kenya kholomo hape ka har'a tube ea pokello, fetisetsa mixtrue ho kholomo, centrifuge ka 12,000 rpm (13,800 × g) bakeng sa 30 - 60 sec.Haeba molumo oa tharollo e tsoakiloeng e le> 700 µL, khutlisetsa kholumo ea adsorption ka har'a tube ea pokello, fetisetsa tharollo e setseng kholumong ea adsorption, le centrifuge ho 12,000 rpm (13,800 × g) bakeng sa 30 - 60 sec.
4. Ts'ebetso e latelang e bua ka mohato oa 5 - 8 oa 08- 1 / lenaneo la ho ntša Gel.
Lisebelisoa
Mefuta e sa tšoaneng ea TAE kapa TBE agarose gel;Lihlahisoa tsa PCR, litsamaiso tsa enzymatic reaction kapa lihlahisoa tse ling tse tala tsa DNA tse fumanoang ka mekhoa e fapaneng.Likhechana tse hlakotsoeng li ne li fapana ho tloha ho70 BP -20 kbps.
Lintlha
Bakeng sa tšebeliso ea lipatlisiso feela.Ha e sebelisoe lits'ebetsong tsa tlhahlobo.
1. Kenya 80 ml ea ethanol ho hlapolla Buffer GW joalokaha ho bontšitsoe tag pele e sebelisoa, boloka mocheso oa kamore.
2. Haeba Buffer GDP e fumaneha habonolo nakong ea polokelo ea mocheso o tlase, e ka behoa mocheso oa kamore nako e itseng pele e sebelisoa.Haeba ho hlokahala, e ka futhumatsoa ka bateng ea metsi ea 37 ℃ ho fihlela moholi o qhibiliha ka ho feletseng, ebe o ka sebelisoa ka mor'a ho kopanya.
3. Beha mocheso oa ho hlapa metsi ho 50 ~ 55 ℃ esale pele.
4. Lenaneong la 08-1 / Gel ea ho ntša Gel mohato oa 1, ho fokotsa boholo ba selae sa gel ho tla fokotsa haholo nako ea ho qhibiliha le ho eketsa katleho ea ho hlaphoheloa (Linearized DNA e bonolo ho hydrolyze ha e tsoela pele ho pepeseha mocheso o phahameng).Se ke oa pepesa gel ea DNA ho UV nako e telele, kaha leseli la ultraviolet le ka baka tšenyo ea DNA.
5. Hlakola gel ho 08- 1 / Gel extraction program mohato oa 2 ka ho feletseng, ho seng joalo ts'ebetso ea ho hlaphoheloa ha DNA e tla ameha haholo.
6. Preheat Elution Buffer kapa ddH2O to 55℃ , e thusang ho ntlafatsa katleho ea DNA elution.Ho kgothaletswa ho boloka DNA ho 2.5 mM Tris-HCl, pH 7.0 - 8.5.
