Obere ihe mmịpụta DNA
Ngwa a na-anabata sistemu nchekwa kacha mma yana teknụzụ nhicha kọlụm silica, nke nwere ike nwetaghachi iberibe DNA 70 bp - 20 kb site na mkpokọta TAE ma ọ bụ TBE agarose gel.Kọlụm mgbasa ozi DNA nwere ike gbasaa DNA nke ọma n'okpuru ọnọdụ nnu dị elu.Na mgbakwunye, ngwa ahụ nwere ike sachapụ iberibe DNA ozugbo site na ngwaahịa PCR, sistemu mmeghachi omume enzymatic ma ọ bụ ngwaahịa DNA crude nke enwetara site na ụzọ ndị ọzọ, wee wepụ adịghị ọcha dị ka protein, ogige ndị ọzọ, ion nnu inorganic na oligonucleotide primers.Ọ nwere ike hụ na enwere ike imecha ọcha n'ime 10-15min.Enwere ike iji DNA dị ọcha mee ihe ozugbo maka ligation, mgbanwe, mgbaze enzyme, transcription in vitro, PCR, usoro, microinjection, wdg.
Ọnọdụ nchekwa
Chekwaa na -15 ~ -25 ℃ ma bufee na ụlọ okpomọkụ.
Ngwa
| Ngwa | (100 rxn) |
| Ihe nchekwa GDP | 80 ml |
| Ihe nchekwa GW | 2 × 20 ml |
| Ihe nchekwa ihe | 20 ml |
| Obere Obere DNA FastPure-G | 100 |
GDP nchekwa:Ihe nchekwa DNA jikọtara.
Ihe nchekwa GW:Ihe nchekwa ịsa;tinye ethanol zuru oke site na olu egosiputara na karama tupu eji ya.
Ihe nchekwa ihe:Elution.
Obere Obere DNA FastPure-G:Ogidi mgbasa ozi DNA.
Tubes mkpokọta 2 ml:Ọkpụkpọ mkpokọta maka nzacha.
Ngwa akwadoro
1.5 ml igba ogwu tubes, zuru ethanol na isopropanol (mgbe DNA iberibe ≤100 bp, tinye 1 olu.
isopropanol ruo 1 olu gel), mmiri ịsa ahụ.
Usoro nnwale
Tinye 80 ml nke ethanol ka igbanwe Buffer GW dị ka egosiri na mkpado tupu eji ya, chekwaa na ụlọ okpomọkụ.
Mechanism
1. Ngwọta mmeghachi omume PCR
Atụmatụ mmịpụta gel: Tinye nha nha nha ihe nchekwa GDP PCR atụmatụ mgbake ngwọta:Tinye ugboro 5 ihe nchekwa olu
2. GDP Gbakọọ olu gel (100 μl bụ 100 mg)
Igbari jel
3. Kpoo ọkụ na 50 ~ 55℃
4. Kechie saa
Tinye 300 μL nke ihe nchekwa GDP*
Tinye 700 μL nke ihe nchekwa GW
Tinye 700 μL nke ihe nchekwa GW
5. Elu
Tinye 20 – 30μL nke Elution Buffer ma ọ bụ mmiri deionized
Ihe ndetu * PCR mmeghachi omume mmiri mgbake na-enweghị usoro a
Usoro mmịpụta gel
1. Mgbe DNA electrophoresis maka fractionating DNA iberibe, wepụ otu straipu nke DNA iberibe si agarose gel n'okpuru UV ìhè.A na-atụ aro ka iji akwụkwọ na-amị amị mkpụrụ iji nweta mmiri mmiri nke gel pụtara ìhè ma belata nha nke iberi gel site na iwepu agarose ọzọ dị ka o kwere mee.Tulee iberi gel (na-enweghị microcentrifuge tube) iji gbakọọ olu ya: Olu nke 100 mg gelslice bụ ihe dịka 100 μL, na-eche na njupụta bụ 1g/ml.
2. Tinye nha nha nhata ihe nchekwa GDP, incubate na 50 ~ 55 ℃ maka 7-10 min (dị ka gel size, gbanwee oge ntinye ruo mgbe gel gbazere kpamkpam).Tụgharịa tube ugboro 2 n'oge incubation.
Δ Mgbakwunye nke 1-3 mpịakọta GDP nke Buffer agaghị emetụta arụmọrụ mgbake DNA.Ọ bụrụ na mpempe DNA ga-enweta <100 bp, ọ dị mkpa ịgbakwunye mpịakọta 3 nke Buffer GDP;mgbe mpempe gel agbazere kpamkpam, tinye 1 olu nke isopropanol ma gwakọta nke ọma, wee gaa n'ihu na nzọụkwụ ọzọ.
3. Centrifuge nkenke iji weta ihe nlele na ala nke tube ahụ, tinye FastPure DNA Mini Columns-G n'ime mkpokọta mkpokọta 2 ml, jiri nlezianya nyefee ngwọta kachasị nke 700 μL otu ugboro a.
oge na ogidi nzacha, centrifuge na 12,000 rpm (13,800 X g) maka 30-60 sec.
4. Tụfuo nzacha ma tinye 300 μL nke Buffer GDP na kọlụm, tinye ya na ụlọ okpomọkụ maka 1 min, centrifuge na 12,000 rpm (13,800 X g) maka 30-60 sec.
5. Tụfuo nzacha ma tinye 700 μL nke Buffer GW (leba anya ma ọ bụrụ na agbakwunyere ethanol zuru oke n'ihu!) Na kọlụm, centrifuge na 12,000 rpm (13,800 X g) maka 30-60 sec.
∆ Biko tinye ihe nchekwa GW n'akụkụ mgbidi kọlụm adsorption, ma ọ bụ tinye Buffer GW mkpuchi azụ wee gwakọta ya n'akụkụ ala maka oge 2 – 3 iji nyere aka kpochapụ nnu na-arapara na mgbidi tube kpamkpam.
6. Tinyegharịa nzọụkwụ 5.
Δ Flushing na Buffer GW ugboro abụọ nwere ike hụ na e wepụrụ nnu kpamkpam ma wepụ mmetụta na nyocha ndị na-esote.
7. Tụfuo nzacha na centrifuge na kọlụm efu na 12,000 rpm (13,800 X g) maka 2 min.
8. Tinye kọlụm n'ime 1.5 ml microcentrifuge tube dị ọcha, tinye 20 - 30 μL nke Elution Buffer n'etiti etiti kọlụm kọlụm, tinye ya maka 2 min, wee centrifuge na 12,000 rpm (13,800 X g) maka 1 min.Tụfuo kọlụm, chekwaa DNA enwetara na -20.
∆ Ịnyefe onye na-ahụ maka nzọụkwụ 8 na kọlụm ka ọ pụta ìhè ọzọ ma na-ekpo ọkụ Elution Buffer na 55 (mgbe DNA mpempe akwụkwọ> 3 kb) nwere ike inye aka ịbawanye arụmọrụ mgbake.
Mmemme mgbake ngwaahịa PCR
Usoro a na-adabara iji sachaa iberibe DNA sitere na ngwaahịa PCR, sistemu mmeghachi omume enzymatic na ngwaahịa DNA ndị ọzọ (gụnyere DNA mkpụrụ ndụ ihe nketa).Ngwọta a nwere ike wepu nucleotides dị iche iche nke ọma, primers, primer dimers, molecules nnu, enzymes na adịghị ọcha ndị ọzọ.
1. Ngwa PCR dị nkenke centrifuge, ngwọta mmeghachi omume enzymatic, na ngwaahịa crude DNA ndị ọzọ.Were pipette tụọ ụda olu ha wee bufee ya na tube 1.5 ml ma ọ bụ 2 ml igba ogwu.Tinye ddH2O ruo mgbe olu ruru 100 μL;ebe maka DNA genomic nwere nlebara anya dị elu, ịgbanye 300 μL na ddH2O ga-enyere aka melite arụmọrụ mgbake.
2. Tinye mpịakọta 5 nke Buffer GDP, gwakọta nke ọma site na ntụgharị ma ọ bụ ntụgharị.Ọ bụrụ na iberibe DNA nke mmasị> 100 bp, ọ dị mkpa ịgbakwunye 1.5 mpịakọta (sample + Buffer GDP) nke ethanol.
3. Tinye kọlụm azụ n'ime tube nchịkọta, nyefee mixtrue na kọlụm, centrifuge na 12,000 rpm (13,800 ×g) maka 30 - 60 sec.Ọ bụrụ na olu nke ihe ngwọta agwakọta bụ> 700 µL, tinye kọlụm adsorption azụ n'ime tube nchịkọta, nyefee ihe ngwọta fọdụrụnụ na kọlụm mgbasa ozi, na centrifuge na 12,000 rpm (13,800 × g) maka 30 - 60 sec.
4. Ọrụ na-esote na-ezo aka na nzọụkwụ 5 - 8 nke 08-1 / gel mmịpụta mmemme.
Ngwa
Ụdị dị iche iche nke TAE ma ọ bụ TBE agarose gel;Ngwaahịa PCR, sistemu mmeghachi omume enzymatic ma ọ bụ ngwaahịa DNA crude ndị ọzọ enwetara site na ụzọ dị iche iche.Iberibe enwetara sitere na70 bp -20 kb.
Ihe ndetu
Maka iji naanị nyocha.Ọ bụghị maka ojiji na usoro nyocha.
1. Tinye 80 ml nke ethanol ka ọ gbanwee Buffer GW dị ka egosiri na mkpado tupu ejiri ya, chekwaa na ụlọ okpomọkụ.
2. Ọ bụrụ na GDP Buffer dị mfe ịmalite n'oge nchekwa okpomọkụ dị ala, enwere ike itinye ya na ụlọ okpomọkụ maka oge tupu ejiri ya.Ọ bụrụ na ọ dị mkpa, ọ nwere ike preheated na a 37 ℃ mmiri bat ruo mgbe precipitate na-kpamkpam agbaze, na mgbe ahụ, ọ nwere ike iji mgbe agwakọta.
3. Tọọ mmiri bat okpomọkụ na 50 ~ 55 ℃ tupu oge eruo.
4. Na 08-1 / gel mmịpụta mmemme nzọụkwụ 1, ibelata size nke gel iberi ga-n'ụzọ dị ukwuu belata dissolving oge na-abawanye mgbake arụmọrụ (Linearized DNA dị mfe hydrolyze mgbe nọgidere na-ekpughe na elu okpomọkụ).Ekpughela gel DNA na UV ruo ogologo oge, n'ihi na ọkụ ultraviolet nwere ike imebi DNA.
5. Gbanyụọ gel na 08- 1 / gel mmịpụta usoro nzọụkwụ 2 kpamkpam, ma ọ bụghị ya, a ga-emetụta ọrụ mgbake DNA nke ọma.
6. Preheat Elution Buffer ma ọ bụ ddH2O ruo 55 ℃ , nke na-enyere aka melite arụmọrụ DNA elution.A na-atụ aro ka ịchekwa DNA na eluent nke 2.5 mM Tris-HCl, pH 7.0 - 8.5.
