DNA Extraction Mini Kit
Iyi kit inotora yakagadziridzwa buffer system uye silica gel column yekuchenesa tekinoroji, iyo inogona kudzoreredza 70 bp - 20 kb DNA zvimedu kubva kwakasiyana siyana kweTAE kana TBE agarose gel.DNA adsorption column inogona kunyanya adsorp DNA pasi pepamusoro-munyu mamiriro.Mukuwedzera, iyo kititi inogona kunatsa zvakananga zvidimbu zveDNA kubva kuPCR zvigadzirwa, enzymatic reaction systems kana crude DNA zvigadzirwa zvakawanikwa nedzimwe nzira, uye kubvisa tsvina yakadai semapuroteni, mamwe makemikari ehupenyu, inorganic salt ions uye oligonucleotide primers.Inogona kuve nechokwadi chekuti kucheneswa kunogona kupedzwa mukati me10-15min.Iyo yakacheneswa DNA inogona kushandiswa zvakananga kune ligation, shanduko, enzyme digestion, in vitro transcription, PCR, sequencing, microinjection, nezvimwe.
Kuchengetedza mamiriro
Chengetedza pa -15 ~ -25 ℃ uye kutakura pamhepo tembiricha.
Zvikamu
| Zvikamu | (100 rxns) |
| Buffer GDP | 80 ml |
| Buffer GW | 2 × 20 ml |
| Elution Buffer | 20 ml |
| FastPure DNA Mini Columns-G | 100 |
Buffer GDP:DNA inosunga buffer.
Buffer GW:Washing buffer;wedzera absolute ethanol nevhoriyamu yakaratidzwa pabhodhoro usati washandisa.
Elution Buffer:Elution.
FastPure DNA Mini Columns-G:DNA adsorption columns.
Machubhu ekuunganidza 2 ml:Machubhu ekuunganidza ekusefa.
Zvinhu Zvakagadzirirwa
1.5 ml sterilized tubes, absolute ethanol uye isopropanol (apo DNA chidimbu ≤100 bp, wedzera 1 vhoriyamu
isopropanol kusvika 1 volume gel), kugeza kwemvura.
Experiment Process
Wedzera 80 ml ye ethanol kuti uwedzere Buffer GW sezvakaratidzwa pa tag usati washandisa, chengeta pakushisa kwekamuri.
Mechanism
1. PCR reaction solution
Gel yekuwedzera chirongwa: Wedzera yakaenzana vhoriyamu Buffer GDP PCR maitiro ekugadzirisa mhinduro chirongwa:Wedzera kashanu vhoriyamu Buffer
2. GDP Verenga huwandu hwegel (100 μndakaenzana ne100 mg)
Dissolve gel
3. Preheat pa50 ~ 55℃
4. Bind Wash
Wedzera 300 μL yeBuffer GDP*
Wedzera 700 μL yeBuffer GW
Wedzera 700 μL yeBuffer GW
5. Elute
Wedzera 20 - 30μL yeElution Buffer kana mvura yakasvibiswa
Notes* PCR reaction fluid kupora pasina iyi nhanho
Gel kubviswa purogiramu
1. Mushure meDNA electrophoresis yekupatsanura zvidimbu zveDNA, bvisa mutsetse mumwechete wechidimbu cheDNA kubva mujeri reagarose pasi pechiedza cheUV.Zvinokurudzirwa kushandisa bepa rinonyudza kuti ritore hunyoro hunoonekwa hwegel uye kuderedza ukuru hwecheki yegel nekubvisa yakawedzera agarose sezvaunokwanisa.Weresa gel slice (isina microcentrifuge chubhu) kuti uverenge huwandu hwayo: Huwandu hwe100 mg gelslice hunenge 100 μL, uchifunga kuti density i1g/ml.
2. Wedzera yakaenzana vhoriyamu Buffer GDP, incubate pa50 ~ 55 ℃ ye 7-10 min (maererano nehukuru hwegel, gadzirisa incubation nguva kusvikira gel yapera zvachose).Pindura chubhu kaviri panguva yekuisa.
Δ Kuwedzerwa kwe1-3 mavhoriyamu eBuffer GDP hakuzopesvedzere DNA kupora kushanda zvakanaka.Kana chidimbu cheDNA chichizodzorerwa <100 bp, 3 mavhoriyamu eBuffer GDP anoda kuwedzerwa;apo gel slice yakanyunguduka zvachose, wedzera 1 vhoriyamu ye isopropanol uye sanganisa zvakakwana, uye ramba uchienda kune danho rinotevera.
3. Centrifuge muchidimbu kuunza sampuli kuzasi kwechubhu, isa FastPure DNA Mini Columns-G muKuunganidzwa Tubes 2 ml, nyatso fambisa mhinduro inokwana 700 μL kamwe chete.
nguva yekusefa makoramu, centrifuge pa12,000 rpm (13,800 X g) ye30-60 sec.
4. Rasa iyo filtrate uye uwedzere 300 μL yeBuffer GDP kumutsara, chengetera pakamuri tembiricha kwe1 min, centrifuge pa12,000 rpm (13,800 X g) ye30-60 sec.
5. Rasa filtrate woisa 700 μL yeBuffer GW (tarisa kana absolute ethanol yawedzerwa kare!) ku column, centrifuge pa 12,000 rpm (13,800 X g) for 30-60 sec.
Δ Ndokumbira uwedzere Buffer GW yakatenderedza adsorption column madziro, kana wedzera Buffer GW yekumashure chivharo uye woisanganisa yakatsikitsira pasi kwe2 - 3 nguva kuti ubatsire zvachose munyu unonamatira kumadziro echubhu.
6. Dzokorora danho rechishanu.
Δ Flushing neBuffer GW kaviri inogona kuve nechokwadi kuti munyu wabviswa zvachose uye kubvisa kukanganiswa kwezviedzo zvinotevera.
7. Rasa filtrate uye centrifuge mbiru isina chinhu pa12,000 rpm (13,800 X g) ye2 min.
8. Isa mbiru muchubhu yakachena 1.5 ml microcentrifuge, wedzera 20 - 30 μL yeElution Buffer kusvika pakati pe column membrane, incubate for 2 min, uye ipapo centrifuge pa 12,000 rpm (13,800 X g) kwe1 min.Rasa chikamu, chengetedza DNA yakawanikwa pa -20 .
Δ Kuendesa iyo supernatant yenhanho 8 kune iyo column kuti iite zvakare uye preheating iyo Elution Buffer ku55 (apo DNA chidimbu> 3 kb) pamwe inobatsira kuwedzera kudzoreredza kugona.
PCR zvigadzirwa zvekudzoreredza chirongwa
Iyi protocol inoshanda kuchenesa zvidimbu zveDNA kubva kuPCR zvigadzirwa, enzymatic reaction system uye zvimwe zvigadzirwa zveDNA crude (kusanganisira genetic DNA).Iyi mhinduro inogona kunyatso bvisa akasiyana nucleotides, maprimers, primer dimers, mamorekuru emunyu, ma enzymes uye kumwe kusvibiswa.
1. Muchidimbu centrifuge PCR zvigadzirwa, enzymatic reaction solution, uye zvimwe DNA crude zvigadzirwa.Ongorora huwandu hwavo nepipette uye uende kune imwe sterilized 1.5 ml kana 2 ml chubhu.Wedzera ddH2O kusvika vhoriyamu kusvika ku100 μL;nepo genomic DNA ine yakakwira concentration, kudilukira ku300 μL neddH2O kuchabatsira kuvandudza kupora.
2. Wedzera 5 mavhoriyamu eBuffer GDP, sanganisa zvakakwana ne inverting kana vortexing.Kana DNA chidimbu chemubereko>100 bp, mamwe 1.5 mavhoriyamu (samples + Buffer GDP) ethanol inoda kuwedzerwa.
3. Isa mbiru kumashure muchubhu yekuunganidza, tumira mixtrue kune mbiru, centrifuge pa 12,000 rpm (13,800 × g) ye 30 - 60 sec.Kana vhoriyamu yemhinduro yakasanganiswa iri> 700 µL, isa adsorption column kumashure muchubhu yekuunganidza, endesa mhinduro yasara kune adsorption column, uye centrifuge pa12,000 rpm (13,800 × g) ye30 - 60 sec.
4. Chiitiko chinotevera chinoreva nhanho 5 - 8 ye08- 1/Gel yekubvisa chirongwa.
Applications
Kusiyana kwakasiyana-siyana kweTAE kana TBE agarose gel;PCR zvigadzirwa, enzymatic reaction system kana zvimwe zvisina kuchena zvigadzirwa zveDNA zvinowanikwa nenzira dzakasiyana.Zvimedu zvakadzorerwa zvakabva70 bp -20 kbps.
Notes
Yekushandisa tsvakurudzo chete.Kwete kushandiswa mukuongorora maitiro.
1. Wedzera 80 ml ye ethanol kuti uderedze Buffer GW sezvinoratidzwa pane tag usati washandisa, chengeta patembiricha yekamuri.
2. Kana iyo Buffer GDP iri nyore kudonha panguva yekudzika-tembiricha yekuchengetedza, inogona kuiswa pakamuri tembiricha kwenguva yakati isati yashandiswa.Kana zvichidikanwa, inogona kupiswa mumvura yekugezesa 37 ℃ kusvika mvura yanyungudika zvachose, uyezve inogona kushandiswa mushure mekusanganiswa.
3. Isa tembiricha yekugezesa mvura kusvika 50 ~55℃ pachine nguva.
4. Mu 08-1 / Gel extraction program danho 1, kuderedza ukuru hwegel slice kuchanyanya kuderedza nguva yekunyunguduka uye kunowedzera kupora kushanda zvakanaka (Linearized DNA iri nyore ku hydrolyze kana ichiramba ichiratidzwa pakupisa kwepamusoro).Usafumure DNA gel kuUV kwenguva yakareba, sezvo ultraviolet mwenje inogona kukonzera kukuvara kweDNA.
5. Dissolve the gel mu 08- 1 / Gel extraction program danho 2 zvachose, kana zvisina kudaro DNA recovery performance ichakanganiswa zvakanyanya.
6. Preheat Elution Buffer kana ddH2O kusvika 55℃, iyo inobatsira kuvandudza DNA elution performance.Zvinokurudzirwa kuchengeta DNA mu 2.5 mM Tris-HCl, pH 7.0 - 8.5.
