DNA Extraction Mini Kit
Cov khoom siv no siv cov txheej txheem tsis zoo thiab silica gel kem purification technology, uas tuaj yeem rov qab tau 70 bp - 20 kb DNA tawg los ntawm ntau qhov ntau ntawm TAE lossis TBE agarose gel.DNA adsorption kab muaj peev xwm tshwj xeeb adsorp DNA nyob rau hauv high-ntsev ntsev.Tsis tas li ntawd, cov khoom siv tuaj yeem ncaj qha purify DNA tawg ntawm cov khoom PCR, cov tshuaj tiv thaiv enzymatic lossis cov khoom siv DNA tsis zoo uas tau txais los ntawm lwm txoj kev, thiab tshem tawm cov impurities xws li cov proteins, lwm cov organic compounds, inorganic ntsev ions thiab oligonucleotide primers.Nws tuaj yeem ua kom huv si tuaj yeem ua tiav hauv 10-15 feeb.Cov DNA purified tuaj yeem siv ncaj qha rau ligation, transformation, enzyme digestion, hauv vitro transcription, PCR, sequencing, microinjection, thiab lwm yam.
Cia tej yam kev mob
Khaws ntawm -15 ~ -25 ℃ thiab thauj hauv chav tsev kub.
Cheebtsam
| Cheebtsam | (100 hli) |
| PIB Buffer | 80ml ib |
| Buffer GW | 2 × 20 ml |
| Elution Buffer | 20 ml ib |
| FastPure DNA Mini Kab-G | 100 |
PIB Buffer:DNA binding tsis.
Buffer GW:Ntxuav tsis;ntxiv ethanol kiag li los ntawm lub ntim qhia ntawm lub raj mis ua ntej siv.
Elution Buffer:Elution.
FastPure DNA Mini Kab-G:DNA adsorption kab.
Collection Tubes 2 ml:Collection tubes rau filtrate.
Cov ntaub ntawv npaj
1.5 ml sterilized raj, absolute ethanol thiab isopropanol (thaum DNA fragment ≤100 bp, ntxiv 1 ntim
isopropanol rau 1 ntim gel), da dej.
Kev sim txheej txheem
Ntxiv 80 ml ntawm ethanol rau dilute Buffer GW raws li qhia ntawm daim ntawv ua ntej siv, khaws cia ntawm chav tsev kub.
Mechanism
1. PCR tshuaj tiv thaiv
Gel extraction scheme: Ntxiv cov ntim sib npaug Buffer GDP PCR cov tshuaj tiv thaiv kev daws teeb meem rov qab:Ntxiv 5 npaug ntawm qhov ntim Buffer
2. GDP xam qhov ntim ntawm gel (100 μl sib npaug 100 mg)
Dissolve gel
3. Preheat ntawm 50 ~ 55℃
4. khi ntxuav
Ntxiv 300 μL ntawm Buffer GDP *
Ntxiv 700 μL ntawm Buffer GW
Ntxiv 700 μL ntawm Buffer GW
5. Elute
Ntxiv 20 - 30μL ntawm Elution Buffer lossis dej deionized
Cov lus ceeb toom * PCR cov tshuaj tiv thaiv cov kua dej rov qab yam tsis muaj cov kauj ruam no
Gel extraction program
1. Tom qab DNA electrophoresis rau fractionating DNA fragments, excise ib kab txaij DNA fragment los ntawm agarose gel nyob rau hauv UV teeb.Nws raug nquahu kom siv cov ntawv nqus dej kom nqus cov dej pom tseeb ntawm cov gel thiab txo qhov loj ntawm cov gel hlais los ntawm kev tshem tawm cov agarose ntxiv raws li koj ua tau.Ntsuas cov gel hlais (tsis muaj microcentrifuge raj) los xam nws cov ntim: Lub ntim ntawm 100 mg gelslice yog kwv yees li 100 μL, piv txwv li qhov ceev yog 1g / ml.
2. Ntxiv qhov sib npaug ntawm qhov tsis sib xws GDP, incubate ntawm 50 ~ 55 ℃ rau 7-10 min (raws li lub gel loj, kho lub sij hawm incubation kom txog thaum lub gel tiav lawm).Invert lub raj 2 zaug thaum lub sij hawm incubation.
Δ Ntxiv ntawm 1-3 ntim ntawm Buffer GDP yuav tsis cuam tshuam DNA rov ua haujlwm zoo.Yog tias cov DNA seem kom rov qab tau <100 bp, 3 ntim ntawm Buffer GDP yuav tsum tau ntxiv;Thaum cov gel hlais tau yaj tag, ntxiv 1 ntim ntawm isopropanol thiab sib tov kom huv si, ces mus rau kauj ruam tom ntej.
3. Centrifuge luv luv kom coj cov qauv mus rau hauv qab ntawm lub raj, ntxig rau FastPure DNA Mini Columns-G rau hauv Collection Tubes 2 ml, ua tib zoo hloov cov tshuaj ntau kawg ntawm 700 μL ib zaug.
lub sij hawm mus rau cov kab pom, centrifuge ntawm 12,000 rpm (13,800 X g) rau 30-60 sec.
4. Tshem tawm cov filtrate thiab ntxiv 300 μL ntawm Buffer GDP rau kem, incubate ntawm chav tsev kub rau 1 min, centrifuge ntawm 12,000 rpm (13,800 X g) rau 30-60 sec.
5. Tshem tawm cov filtrate thiab ntxiv 700 μL ntawm Buffer GW (xyuas seb puas muaj ethanol tau ntxiv ua ntej!) rau hauv kem, centrifuge ntawm 12,000 rpm (13,800 X g) rau 30-60 sec.
Δ Thov ntxiv Buffer GW nyob ib ncig ntawm phab ntsa adsorption kem, lossis ntxiv Buffer GW rov qab npog thiab sib xyaw ua ke rau 2 - 3 zaug los pab tshem tawm cov ntsev kom tag rau lub raj phab ntsa.
6. Rov ua kauj ruam 5.
Δ Flushing nrog Buffer GW ob zaug tuaj yeem xyuas kom meej tias cov ntsev raug tshem tawm tag nrho thiab tshem tawm qhov cuam tshuam ntawm kev sim tom ntej.
7. Tshem tawm cov filtrate thiab centrifuge lub khoob ntawm 12,000 rpm (13,800 X g) rau 2 min.
8. Ntxig lub kem mus rau hauv ib qho huv 1.5 ml microcentrifuge raj, ntxiv 20 - 30 μL ntawm Elution Buffer rau hauv nruab nrab ntawm kab kem, incubate rau 2 min, thiab ces centrifuge ntawm 12,000 rpm (13,800 X g) rau 1 min.Tshem tawm cov kab, khaws cov DNA tau ntawm -20 .
Δ Hloov lub supernatant ntawm kauj ruam 8 mus rau kem kom elute dua thiab preheating lub Elution Buffer rau 55 (thaum DNA fragment> 3 kb) tej zaum yuav pab tau kom lub rov qab efficiency.
PCR cov khoom siv rov ua haujlwm
Cov txheej txheem no siv tau los ua kom huv DNA tawg ntawm cov khoom PCR, cov tshuaj tiv thaiv enzymatic thiab lwm yam khoom siv DNA (xws li caj ces DNA).Cov tshuaj no tuaj yeem tshem tawm ntau yam nucleotides, primers, primer dimers, ntsev molecules, enzymes thiab lwm yam impurities.
1. Luv luv centrifuge PCR cov khoom, enzymatic cov tshuaj tiv thaiv, thiab lwm yam DNA crude khoom.Kwv yees lawv cov ntim nrog pipette thiab hloov mus rau sterilized 1.5 ml lossis 2 ml raj.Ntxiv ddH2O kom txog thaum lub ntim mus txog 100 μL;thaum rau genomic DNA nrog siab concentration, diluting rau 300 μL nrog ddH2O yuav pab txhim kho kev rov qab efficiency.
2. Ntxiv 5 ntim ntawm Buffer GDP, sib tov kom huv si los ntawm inverting los yog vortexing.Yog tias DNA tawg ntawm kev txaus siab> 100 bp, ntxiv 1.5 ntim (samples + Buffer GDP) ntawm ethanol yuav tsum tau ntxiv.
3. Ntxig lub kem rov qab rau hauv lub raj sau, hloov cov mixtrue mus rau kem, centrifuge ntawm 12,000 rpm (13,800 × g) rau 30 - 60 sec.Yog tias qhov ntim ntawm cov tshuaj sib xyaw yog> 700 µL, muab cov adsorption kem rov qab rau hauv lub raj sau, hloov cov tshuaj ntxiv mus rau sab adsorption, thiab centrifuge ntawm 12,000 rpm (13,800 × g) rau 30 - 60 sec.
4. Qhov kev ua tau zoo tom ntej yog hais txog qib 5 – 8 ntawm 08- 1/Gel extraction program.
Daim ntawv thov
Ntau qhov ntau ntawm TAE lossis TBE agarose gel;PCR cov khoom, cov tshuaj tiv thaiv enzymatic lossis lwm yam khoom siv DNA tsis zoo tau txais los ntawm ntau txoj hauv kev.Rov qab fragments ranged los ntawm70 bp - 20 kb.
Sau ntawv
Rau kev tshawb fawb siv xwb.Tsis siv rau hauv cov txheej txheem kuaj mob.
1. Ntxiv 80 ml ntawm ethanol rau dilute Buffer GW raws li qhia rau ntawm daim ntawv ua ntej siv, khaws cia rau hauv chav tsev kub.
2. Yog tias Buffer GDP yog ib qho yooj yim los nag thaum lub sij hawm cia txias, nws tuaj yeem muab tso rau hauv chav sov rau lub sijhawm ua ntej siv.Yog tias tsim nyog, nws tuaj yeem ua rau preheated hauv 37 ℃ dej da dej kom txog thaum cov dej nag tau yaj tag, thiab tom qab ntawd nws tuaj yeem siv tom qab sib tov.
3. Teem lub da dej kub rau 50 ~ 55 ℃ ua ntej.
4. Nyob rau hauv 08-1 / Gel extraction program kauj ruam 1, txo qhov luaj li cas ntawm cov gel hlais yuav txo tau lub sij hawm dissolving thiab ua rau kom rov ua hauj lwm zoo (Linearized DNA yog yooj yim rau hydrolyze thaum pheej raug kub kub).Tsis txhob nthuav tawm DNA gel rau UV ntev, vim tias lub teeb ultraviolet tuaj yeem ua rau DNA puas.
5. Dissolve gel nyob rau hauv 08- 1 / Gel extraction program kauj ruam 2 kiag li, txwv tsis pub cov DNA rov ua hauj lwm yuav raug cuam tshuam loj heev.
6. Preheat Elution Buffer los yog ddH2O rau 55 ℃, uas yog pab txhim kho DNA elution efficiency.Nws raug nquahu kom khaws DNA hauv eluent ntawm 2.5 mM Tris-HCl, pH 7.0 - 8.5.
