DNA Extraction Mini Kit
Chidachi chimagwiritsa ntchito makina oyeretsera a silika ndi silika gel oyeretsa, omwe amatha kupezanso zidutswa za 70 bp - 20 kb DNA kuchokera kumagulu osiyanasiyana a TAE kapena TBE agarose gel.DNA adsorption column imatha kutsatsa mwapadera DNA pansi pa mchere wambiri.Kuphatikiza apo, zidazi zimatha kuyeretsa mwachindunji zidutswa za DNA kuchokera kuzinthu za PCR, ma enzymatic reaction system kapena zinthu zopanda DNA zomwe zimapezedwa ndi njira zina, ndikuchotsa zonyansa monga mapuloteni, mankhwala ena achilengedwe, ayoni amchere amchere ndi oligonucleotide primers.Zitha kuonetsetsa kuti kuyeretsedwa kutha kutha mkati mwa 10-15min.DNA yoyeretsedwa ingagwiritsidwe ntchito mwachindunji kulumikiza, kusinthika, kugaya kwa enzyme, kulembedwa mu vitro, PCR, kutsatizana, microinjection, etc.
Zosungirako
Sungani pa -15 ~ -25 ℃ ndi zoyendera kutentha firiji.
Zigawo
| Zigawo | (100 rxns) |
| Kuchepetsa GDP | 80 ml pa |
| Mtengo wa GW | 2 × 20 ml |
| Elution Buffer | 20 ml |
| FastPure DNA Mini Columns-G | 100 |
Kuchepetsa GDP:DNA yomangiriza buffer.
Mtengo wa GW:Kutsuka bafa;onjezani Mowa wathunthu ndi voliyumu yomwe yawonetsedwa pabotolo musanagwiritse ntchito.
Elution Buffer:Elution.
FastPure DNA Mini Columns-G:DNA adsorption columns.
Machubu osonkhanitsira 2 ml:Machubu osonkhanitsira osefera.
Zida Zokonzekera
1.5 ml machubu osabala, ethanol mtheradi ndi isopropanol (pamene DNA fragment ≤100 bp, onjezani voliyumu imodzi
isopropanol ku 1 voliyumu gel osakaniza), osamba madzi.
Njira Yoyesera
Onjezani 80 ml ya ethanol kuti muchepetse Buffer GW monga momwe zasonyezedwera pa tag musanagwiritse ntchito, sungani kutentha.
Njira
1. PCR reaction solution
Chiwembu chochotsa gel osakaniza: Onjezani voliyumu yofanana ya Buffer GDP PCR reaction solution scheme:Onjezani kasanu kuchuluka kwa voliyumu ya Buffer
2. GDP Werengani kuchuluka kwa gel osakaniza (100 μl ndi 100 mg)
Sungunulani gel osakaniza
3. Preheat pa 50 ~ 55℃
4. Amanga Sambani
Onjezani 300 μL ya Buffer GDP *
Onjezani 700 μL ya Buffer GW
Onjezani 700 μL ya Buffer GW
5. Elute
Onjezani 20 - 30μL ya Elution Buffer kapena madzi opangidwa ndi deionized
Zolemba* PCR reaction fluid kuchira popanda sitepe iyi
Pulogalamu yochotsa gel osakaniza
1. Pambuyo pa DNA electrophoresis pogawa zidutswa za DNA, chotsani mzere umodzi wa chidutswa cha DNA kuchokera ku gel agarose pansi pa kuwala kwa UV.Ndibwino kuti mugwiritse ntchito pepala loyamwa kuti mutenge chinyezi cha gel ndikuchepetsa kukula kwa kagawo ka gel osakaniza pochotsa agarose owonjezera momwe mungathere.Yezerani kagawo ka gel osakaniza (popanda microcentrifuge chubu) kuti muwerenge kuchuluka kwake: Kuchuluka kwa 100 mg gelslice ndi pafupifupi 100 μL, poganiza kuti makulidwe ake ndi 1g/ml.
2. Onjezani voliyumu yofanana ya Buffer GDP, sungani pa 50 ~ 55 ℃ kwa 7-10 min (malinga ndi kukula kwa gel, sinthani nthawi yowonongeka mpaka gel osakaniza atasungunuka kwathunthu).Pindutsani chubu ka 2 pa nthawi yoyamwitsa.
Δ Kuphatikizika kwa ma voliyumu 1-3 a Buffer GDP sikungakhudze kuchira kwa DNA.Ngati chidutswa cha DNA chibwezeretsedwe <100 bp, ma voliyumu 3 a Buffer GDP ayenera kuwonjezeredwa;pamene gawo la gel lasungunuka kwathunthu, onjezerani 1 voliyumu ya isopropanol ndikusakaniza bwino, kenaka pitirizani ku sitepe yotsatira.
3. Centrifuge mwachidule kubweretsa chitsanzo pansi pa chubu, ikani FastPure DNA Mini Columns-G mu Machubu Osonkhanitsa 2 ml, tumizani mosamala yankho la 700 μL kamodzi kamodzi.
nthawi yofikira pazipilala zosefera, centrifuge pa 12,000 rpm (13,800 X g) kwa masekondi 30-60.
4. Tayani zosefera ndikuwonjezera 300 μL ya Buffer GDP kugawo, ikani kutentha kwa mphindi imodzi, centrifuge pa 12,000 rpm (13,800 X g) kwa 30-60 sec.
5. Tayani kusefera ndikuwonjezera 700 μL ya Buffer GW (onani ngati ethanol mtheradi wawonjezedwa pasadakhale!) ku gawoli, centrifuge pa 12,000 rpm (13,800 X g) kwa 30-60 sec.
Δ Chonde onjezani Buffer GW mozungulira khoma la adsorption, kapena onjezani Buffer GW yakumbuyo ndikusakaniza mozondoka kwa 2 - 3 nthawi kuti muthandizire kutulutsa mchere womwe umamatira ku khoma la chubu.
6. Bwerezani sitepe 5.
Δ Flushing ndi Buffer GW kawiri kungathe kuonetsetsa kuti mcherewo wachotsedwa kwathunthu ndikuchotsa zotsatira za mayesero otsatila.
7. Tayani filtrate ndi centrifuge ndime yopanda kanthu pa 12,000 rpm (13,800 X g) kwa 2 min.
8. Lowetsani ndime mu chubu choyera cha 1.5 ml microcentrifuge, onjezerani 20 - 30 μL ya Elution Buffer pakatikati pa nembanemba ya ndime, incubate kwa 2 min, ndiyeno centrifuge pa 12,000 rpm (13,800 X g) kwa mphindi imodzi.Tayani ndime, sungani DNA yopezeka pa -20 .
Δ Kusamutsa wapamwamba kwambiri wa sitepe 8 ku gawoli kuti atulutsenso ndikutenthetsa Elution Buffer ku 55 (pamene DNA fragment> 3 kb) mwina ingathandize kuwonjezera kuchira.
Pulogalamu yobwezeretsa zinthu za PCR
Protocol iyi imagwira ntchito poyeretsa zidutswa za DNA kuchokera kuzinthu za PCR, enzymatic reaction system ndi zinthu zina zopanda DNA (kuphatikiza DNA).Njirayi imatha kuchotsa bwino ma nucleotides, zoyambira, ma dimers oyambira, mamolekyu amchere, michere ndi zonyansa zina.
1. Mwachidule centrifuge PCR mankhwala, enzymatic reaction solution, ndi zina DNA crude mankhwala.Yerekezerani kuchuluka kwake ndi pipette ndikusamutsira ku chubu chosawilitsidwa cha 1.5 ml kapena 2 ml.Onjezani ddH2O mpaka voliyumu mpaka 100 μL;pomwe DNA ya genomic yokhala ndi chidwi kwambiri, kuchepetsedwa mpaka 300 μL ndi ddH2O kumathandizira kuchira bwino.
2. Onjezani ma voliyumu 5 a Buffer GDP, sakanizani bwino ndi inverting kapena vortexing.Ngati DNA fragment of interest>100 bp, 1.5 voliyumu yowonjezera (zitsanzo + Buffer GDP) ya ethanol iyenera kuwonjezeredwa.
3. Lowetsani ndime mmbuyo mu chubu chosonkhanitsira, tumizani mixtrue ku ndime, centrifuge pa 12,000 rpm (13,800 × g) kwa 30 - 60 sec.Ngati voliyumu ya yankho losakanikirana ndi> 700 µL, ikani gawo la adsorption mu chubu chosonkhanitsira, tumizani njira yotsalayo ku gawo la adsorption, ndi centrifuge pa 12,000 rpm (13,800 × g) kwa 30 - 60 sec.
4. Ntchito yotsatira ikunena za sitepe 5 - 8 ya 08- 1 / Gel yotulutsa pulogalamu.
Mapulogalamu
Zosiyanasiyana za TAE kapena TBE agarose gel;Zinthu za PCR, ma enzymatic reaction systems kapena zinthu zina za DNA zamwano zopezedwa ndi njira zosiyanasiyana.Zidutswa zobwezeretsedwa zinali zochokera70 bp -20 kbps.
Zolemba
Kugwiritsa ntchito kafukufuku kokha.Osagwiritsidwa ntchito pozindikira matenda.
1. Onjezerani 80 ml ya ethanol kuti muchepetse Buffer GW monga momwe tawonetsera pa tag musanagwiritse ntchito, sungani kutentha.
2. Ngati Buffer GDP ndi yosavuta kugwa panthawi yosungirako kutentha, ikhoza kuikidwa pa kutentha kwa nthawi yaitali musanagwiritse ntchito.Ngati n'koyenera, akhoza preheated mu osamba madzi 37 ℃ mpaka mpweya kusungunuka kwathunthu, ndiyeno angagwiritsidwe ntchito pambuyo kusakaniza.
3. Ikani kutentha kwa madzi osamba ku 50 ~ 55 ℃ pasadakhale.
4. Mu 08-1 / gel osakaniza pulogalamu sitepe 1, kuchepetsa kukula kwa gel osakaniza kagawo kudzachepetsa kwambiri Kutha nthawi ndi kumawonjezera kuchira bwino (Linearized DNA mosavuta hydrolyze pamene mosalekeza poyera kutentha).Osawonetsa gel wa DNA ku UV kwa nthawi yayitali, chifukwa kuwala kwa ultraviolet kumatha kuwononga DNA.
5. Sungunulani gel osakaniza mu 08- 1 / Gel m'zigawo pulogalamu sitepe 2 kwathunthu, apo ayi DNA kuchira bwino adzakhudzidwa kwambiri.
6. Preheat Elution Buffer kapena ddH2O to 55℃ , zomwe zimathandiza kupititsa patsogolo kutulutsa kwa DNA.Ndikoyenera kusunga DNA mu 2.5 mM Tris-HCl, pH 7.0 - 8.5.
