Qalabka yar ee Soo saarista DNA
Xirmadani waxa ay qabataa habka wax-ka-qabashada ee la hagaajiyay iyo tignoolajiyada silica gel column, kaas oo ka soo kaban kara 70 bp – 20 kb jajabyada DNA-da ee kala duwan ee TAE ama TBE agarose gel.Tiirka xayeysiinta DNA-ga wuxuu si gaar ah u xayeysiin karaa DNA-da xaalad milixeed sare leh.Intaa waxaa dheer, qalabku wuxuu si toos ah u nadiifin karaa jajabyada DNA-da ee alaabta PCR, hababka falcelinta enzymatic ama alaabada DNA-da cayriin ee laga helo habab kale, oo ka saara wasakhda sida borotiinka, xeryahooda kale ee dabiiciga ah, ions cusbada ah iyo oligonucleotide primers.Waxay xaqiijin kartaa in nadiifinta lagu dhammeeyo 10-15 daqiiqo gudahood.DNA-da la safeeyey waxaa si toos ah loogu isticmaali karaa ligation, beddelaad, dheefshiidka enzyme, qoraalka in vitro, PCR, isku xigxiga, microinjection, iwm.
Xaaladaha kaydinta
Ku kaydi at -15 ~ -25 ℃ kuna qaado heerkulka qolka.
Qaybaha
| Qaybaha | (100 rxn) |
| Waxqabadka GDP | 80 ml |
| Buffer GW | 2 × 20 ml |
| Elution Buffer | 20 ml |
| FastPure DNA Mini Columns-G | 100 |
Wax-soo-saarka kaydka ah:DNA xidhidhiyaha
Buffer GW:Bakhaar lagu dhaqo;ku dar ethanol dhamaystiran mugga la tilmaamay ee dhalada ka hor inta aan la isticmaalin.
Ilbaxnimada Elution:Ilbaxnimo.
FastPure DNA Mini Columns-G:Tiirarka adsorption DNA.
Tuubooyinka Aruurinta 2 ml:Tuubooyinka aruurinta sifeynta
Agabka la diyaariyay
1.5 ml tuubooyinka jeermis dilay, ethanol buuxda iyo isopropanol (marka jajabka DNA ≤100 bp, ku dar 1 mug
isopropanol ilaa 1 mugga jel), qubeyska biyaha.
Habka tijaabada
Ku dar 80 ml oo ethanol ah si aad u milo Buffer GW sida ku cad summada kahor isticmaalka, ku kaydi heerkulka qolka.
Farsamaynta
1. Xalka falcelinta PCR
Nidaamka soo saarista jel:Ku dar mugga siman ee wax-ka-qabashada GDP PCR nidaamka soo kabashada:Ku dar 5 jeer mugga kaydinta
2. GDP Xisaabi mugga jel (100 μl waxay la mid tahay 100 mg)
milanka jel
3. Kuleylka 50 ~ 55℃
4. Dhaq dhaqidda
Ku dar 300 μL ee GDP kaydinta*
Ku dar 700 μL ee Buffer GW
Ku dar 700 μL ee Buffer GW
5. Elute
Ku dar 20 - 30μL oo ah Elution Buffer ama biyo la miiray
Xusuusin * PCR falcelinta dareeraha soo kabashada la'aanteed tallaabadan
barnaamijka soo saarista jel
1. Ka dib DNA electrophoresis ee jajabinta jajabyada DNA-da, ka saar xariijimaha keli ah ee jajabka DNA-ga agarose ee hoosta iftiinka UV.Waxaa lagugula talinayaa inaad isticmaasho warqad nuugista si aad u nuugo qoyaanka muuqda ee jel oo aad yareyso xajmiga jelka adigoo ka saaraya agarose dheeraad ah intii suurtagal ah.Miisaanka jel-jeexa (la'aanteed tuubada microcentrifuge) si aad u xisaabiso muggeeda: Mugga 100 mg gelslice waa ku dhawaad 100 μL, iyada oo loo maleynayo in cufnaanta ay tahay 1g/ml.
2. Ku dar mugga siman ee Buffer GDP, ku dheji 50 ~ 55 ℃ 7-10 min (sida ku cad cabbirka jel, hagaajiso wakhtiga soo-galka ilaa jelku si buuxda u milmo).U rog tubbada 2 jeer inta lagu jiro kuleejka.
∆ Ku darida 1-3 mugga ee Buffer GDP ma saameyn doonto waxtarka soo kabashada DNA.Haddii jajabka DNA-da ee la soo celinayo <100 bp, 3 mug ee Buffer GDP ayaa u baahan in lagu daro;Marka jelku si buuxda u milmo, ku dar 1 mugga isopropanol oo si fiican u walaaq, ka dibna sii wad tallaabada xigta.
3. Centrifuge si kooban si aad muunadda u keento tuubada hoose, geli FastPure DNA Mini Columns-G tubbada aruurinta 2 ml, si taxadar leh ugu wareeji xalka ugu badnaan 700 μL hal mar
wakhtiga tiirarka sifaynta, centrifuge at 12,000 rpm (13,800 X g) 30-60 sek.
4. Tuur shaandhada oo ku dar 300 μL ee Buffer GDP tiirka, ku dhex geli heerkulka qolka 1 daqiiqo, sentrifuge at 12,000 rpm (13,800 X g) 30-60 sek.
5. Tuur shaandhada oo ku dar 700 μL ee Buffer GW (hubi haddii ethanol dhammaystiran horay loogu daray!) tiirka, centrifuge at 12,000 rpm (13,800 X g) 30-60 seken.
∆ Fadlan ku dar Buffer GW agagaarka gidaarka tiirka xayeysiinta, ama ku dar Buffer GW daboolka dambe oo ku qas kor ilaa 2 – 3 jeer si aad si buuxda uga caawiso nadiifinta milixda ku dheggan gidaarka tuubada.
6. Ku celi talaabada 5.
∆ Ku-qulqulka Buffer GW laba jeer waxay hubin kartaa in milixda gebi ahaanba meesha laga saaray oo ay meesha ka saarto saamaynta tijaabooyinka xiga.
7. Tuur shaandhaynta oo badhkii tiirka madhan 12,000 rpm (13,800 X g) 2 daqiiqo.
8. Geli tiirka 1.5 ml tuubo microcentrifuge nadiif ah, ku dar 20 - 30 μL ee Elution Buffer bartamaha xuubka tiirka, ku rid 2 daqiiqo, ka dibna sentifuge at 12,000 rpm (13,800 X g) 1 daqiiqo.Tuur tiirka, ku kaydi DNA-ga la helay at -20 .
∆ U wareejinta heerka sare ee tiirka 8 si uu mar kale u faafo oo u kululeeyo Elution Buffer ilaa 55 (marka jajabka DNA>3 kb) waxaa laga yaabaa inay ku caawiso kordhinta waxtarka soo kabashada.
PCR barnaamijka soo kabashada alaabta
Habkani waa mid lagu dabaqi karo si loo nadiifiyo jajabyada DNA-da ee alaabta PCR, habka falcelinta enzymatic iyo alaabta kale ee DNA-ga ah (oo ay ku jiraan DNA-da hidde-socodka).Xalkani wuxuu si wax ku ool ah uga saari karaa nucleotide kala duwan, asal-u-dhigayaasha, dimers-ka-soo-saarka, molecules milixda, enzymes iyo wasakh kale.
1. Si kooban centrifuge alaabta PCR, xal falcelinta enzymatic, iyo alaabta kale ee cayriin DNA.Ku qiyaas cabbirkooda pipette oo ku wareeji tuubo 1.5 ml oo jeermis ah ama 2 ml ah.Ku dar ddH2O ilaa mugga ilaa 100 μL;halka DNA-da genomic ee leh feejignaan sare, lagu qasi karo 300 μL ddH2O waxay gacan ka geysan doontaa hagaajinta waxtarka soo kabashada.
2. Ku dar 5 mujallad oo ah Buffer GDP, si fiican isku qas adigoo roganaya ama gariiraya.Haddii jajabka DNA-da ee xiisaha> 100 bp, 1.5 mug dheeraad ah (muunado + Buffer GDP) ee ethanol ayaa loo baahan yahay in lagu daro.
3. Tiirka dib ugu geli tuubada ururinta, ku wareeji isku darka tiirka, sentrifuge at 12,000 rpm (13,800 ×g) 30 - 60 sek.Haddii mugga xalka isku dhafan uu yahay> 700 µL, ku celi tiirka xayeysiinta ee ku celi tuubada aruurinta, ku wareeji xalka haray tiirka xayeysiinta, oo centrifuge 12,000 rpm (13,800 × g) 30 - 60 sek.
4. Waxqabadka soo socdaa waxa uu tilmaamayaa tillaabada 5 – 8 ee 08- 1/barnaamijka soo saarista Gel.
Codsiyada
Xeelado kala duwan oo TAE ama TBE agarose gel;Alaabooyinka PCR, hababka falcelinta enzymatic ama alaabada kale ee DNA-da cayriin ee lagu helay habab kala duwan.Qaybo la soo kabsaday ayaa ka koobnaa70 bp -20 kb.
Xusuusin
Isticmaalka cilmi-baarista kaliya.Looma isticmaalo hababka ogaanshaha.
1. Ku dar 80 ml oo ethanol ah si aad u milo Buffer GW sida ku cad summada ka hor isticmaalka, ku kaydi heerkulka qolka.
2. Haddii Buffer GDP ay fududahay in la soo da'o inta lagu jiro kaydinta heerkulka hooseeya, waxaa lagu dhejin karaa heerkulka qolka muddo wakhti ah kahor isticmaalka.Haddii loo baahdo, waxaa lagu sii kululayn karaa biyaha qubeyska 37 ℃ ilaa uu roobku si buuxda u milmo, ka dibna waxaa la isticmaali karaa ka dib marka la isku daro.
3. Horay u dhig heerkulka qubayska 50 ~ 55℃.
4. In 08-1/Barnaamijka soo saarista jel tallaabada 1, yareeynta xajmiga jel jel waxa ay si weyn u dhimi doontaa wakhtiga kala dirida waxayna kordhisaa waxtarka soo kabashadaHa u soo bandhigin jel DNA UV muddo dheer, sababtoo ah iftiinka ultraviolet wuxuu sababi karaa waxyeelo DNA.
5. Ku milanka jel ee 08-1/Barnaamijka soo saarista jel tallaabada 2 si buuxda, haddii kale waxtarka soo kabashada DNA-da ayaa si xun u saamayn doonta.
6. Preheat Elution Buffer ama ddH2O ilaa 55 ℃, kaas oo waxtar u leh hagaajinta waxtarka sare ee DNA.Waxaa lagu talinayaa in lagu keydiyo DNA-ga 2.5 mM Tris-HCl, pH 7.0 - 8.5.
