Superstart qPCR Premix gbakwunyere-UNG
Mpempe nwamba: HCB5071E
Superstart qPCR Premix gbakwunyere-UNG bụ reagent pụrụ iche emebere maka ezigbo mmeghachi omume PCR na nha nha site na iji nchọpụta dabere na nyocha, emepụtara maka usoro lyophilization.Ọ nwere ihe ọhụrụ hotstart Taq gbakwunyere (DG), nke nwere ọrụ enzyme Taq nke etinyere n'ime ụlọ okpomọkụ, na-egbochi nkwalite nke na-abụghị nke akọwapụtara nke ọma site na nkwụsị nke primer na-abụghị nke annealing ma ọ bụ primer dimer n'okpuru ọnọdụ okpomọkụ dị ala, si otú a na-eme ka ọ dịkwuo mma. nkọwa nke mmeghachi omume mmụba.Reagent a na-eji ihe nchekwa qPCR emepụtara nke ọma yana sistemu mgbochi UNG/dUTP iji nweta mmalite ọkụ ngwa ngwa, na-eme ka arụmọrụ na nghọta nke mmeghachi omume qPCR dịkwuo mma.Ọ nwere ike nweta akụkụ dị mma nke ọkọlọtọ n'ọtụtụ mpaghara ọnụọgụ ma rụọ ọrụ nke ọma, na-egbochi nkwalite ụgha ụgha nke ngwaahịa PCR fọdụrụnụ ma ọ bụ mmetọ ikuku.Nke a reagent dakọtara na ọtụtụ fluorescent quantitative PCR ngwa sitere na ndị nrụpụta dị ka Applied Biosystems, Eppendorf, Bio-Rad na Roche wdg, ma na-egosipụta nkwụsi ike dị mma n'ụdị lyophilized.
Ngwakọta reagent
1. 5× HotstartPremix plus-UNG (Mg2+n'efu) (DG)
2. 250 mmMgCl2
3. 4 × lyoprotectant (nhọrọ)
Ọnọdụ Nchekwa
nchekwa ogologo oge na -20 ℃;enwere ike ịchekwa na 4 ℃ ruo ọnwa 3.Mix ọma tupu ojiji nazere oyi ugboro ugboro na agbaze.
Protocol ịgba ígwè
| Usoro | Okpomọkụ | Oge | okirikiri |
| mgbaze | 50 ℃ | 2 min | 1 |
| Polymerase ịgbalite | 95 ℃ | Nkeji 1 ~ 5 | 1 |
| Denature | 95 ℃ | 10 ~ 20 nkeji | 40-50 |
| Mkpuchi na ndọtị | 56-64 ℃ | 20 ~ 60 nkeji | 40-50 |
qPCR Liquid Reaction Syazuokokoosisi Nkwadebe
|
Ihe mejupụtara |
25µL olu |
50µL olu |
Ntụkwasị obi ikpeazụ |
| 5× HotstartPremix gbakwunyere-UNG(Mg2+n'efu) (DG) | 5µL | 10µL | 1× |
| 250mMgCl2 | 0.45µL | 0.9µL | 4.5 mm |
| 4 × lyoprotectant1 | 6.25µL | 12.5µL | 1× |
| Ngwakọta 25 × Primer-Probe Mix2 | 1µL | 2µL | 1× |
| DNA template3 | —— | —— | —— |
| ddH2O | Maka 25µL | Maka 50µL | —— |
1. Nkwenye ikpeazụ nke 0.2μM maka primers na-arụpụtakarị ihe dị mma;mgbe arụmọrụ mmeghachi omume na-adịghị mma, dozie ntinye nke primer n'ime oke nke 0.2-1μM dị ka ọ dị mkpa.A na-edobe mkpokọta nyocha n'ime oke 0.1-0.3μM site na nnwale gradient iji chọta ngwakọta kacha mma.
2. Nọmba nnomi nke mkpụrụ ndụ ihe mgbaru ọsọ dị na ụdị ndebiri dị iche iche dịgasị iche iche;ọ bụrụ na ọ dị mkpa, enwere ike ịme dilution gradient iji chọpụta ego mgbakwunye template kacha mma.
3. Usoro a nwere ike lyophilized;mgbe ndị ahịa na-eji usoro a na-enweghị ihicha-ihicha chọrọ, 4 × lyoprotectant nwere ike họrọ nhọrọ; ma ọ bụrụ na enwere ngwaahịa akpọnwụ akpọnwụ achọrọ, n'oge nnabata mmiri reagents ogbo ngwaahịa arụmọrụ, ọ ga-agbakwunye 4 × lyoprotectant iji hụ na ọ dabara na akụrụngwa sistemụ lyophilized. na mmetụta.
Mgbe eji usorod maka ifriizi-ihicha, dozie ya usoro as na-eso:
| Ihe mejupụtara | 25µL Sistemụ mmeghachi omume |
| 5 × HotstartPremix gbakwunyere-UNG (Mg2+n'efu) (DG) | 5µL |
| 250mMgCl2 | 0.45µL |
| 4 × lyoprotectant | 6.25µL |
| Ngwakọta 25 × Primer-Probe Mix | 1µL |
| ddH2O | Maka 18-20µL |
* Ọ bụrụ na achọrọ sistemu ndị ọzọ maka ihicha friza, biko jụọ iche.
Usoro Lyophilizationss
| Usoro | Okpomọkụ | Oge | Ọnọdụ | Nrụgide |
| Na-ebu ụzọ kefriza | 4 ℃ | 30 min | Jide |
1 atm |
| -50 ℃ | 60 min | Na-ajụ oyi | ||
| -50 ℃ | 180 min | Jide | ||
| Ihicha nke izizi | -30 ℃ | 60 min | kpo oku |
Oke Vacuum |
| -30 ℃ | 70 min | Jide | ||
| ihicha nke abụọ | 25 ℃ | 60 min | kpo oku |
Oke Vacuum |
| 25 ℃ | 300 min | Jide |
2. Usoro lyophilization dị n'elu bụ maka ntụnye aka naanị.Ụdị ngwaahịa dị iche iche na ndị na-ehicha friza dị iche iche nwere parampat dị iche iche, yabụ enwere ike ime mgbanwe dịka ọ dị n'ezieọnọdụ n'oge eji.
3. Usoro lyophilization dị iche iche nwere ike ịdị mma maka nha dị iche iche nke lyophilizedngwaahịa, n'ihi ya, a ga-emerịrị nkwado nyocha zuru oke mgbe ejiri ya mee ihe maka mmepụta nnukwu.
Ntuziaka maka iji lyophilized ntụ ntụ
1. Na nkenke centrifuge ntụ ntụ lyophilized;
2. Tinye template nucleic acid na ntụ ntụ lyophilized wee tinye mmiri ruo 25µL;
3. Gwakọta nke ọma site na centrifugation na-agba ọsọ na igwe.
Njikwa ogo:
1. Nnwale ọrụ: uche, nkọwapụta, nrụpụta nke qPCR.
2. Ọ dịghị ọrụ nuclease exogenous, ọ dịghị exogenous endo / exonuclease mmetọ.
Ozi nka na ụzụ:
1. Superstart qPCR Premix plus-UNG na-eji enzyme na-ekpo ọkụ na-amalite ngwa ngwa nke na-enyere aka ịmalite ọkụ ngwa ngwa n'ime 1 ~ 5 nkeji;site na nhazi ihe nchekwa pụrụ iche ọ dabara maka mmeghachi omume PCR quantitative multiplex.
2. Ọ nwere elu Specific nke budata mma uche nke fluorescence quantitative PCR njedebe nchọpụta,eme amplification curves normalization, fluorescence uru nweta doro anya mmelite na ala ịta ndebiri, adabara dị ka elu uche fluorescence quantitative PCR nchọpụta reagents.
3. Maka primers nwere okpomọkụ na-egbu mgbu ma ọ bụ karịa 200bp iberibe, a na-atụ aro usoro nzọụkwụ 3.
4. The itinye n'ọrụ arụmọrụ nke dUTP na uche na UNG enzyme dị iche iche maka dị iche iche lekwasịrị mkpụrụ ndụ ihe nketa, otú ọ bụrụ na iji UNG usoro na-eduga mbelata na nchọpụta uche, usoro mmeghachi omume kwesịrị gbanwee na kacha.Ọ bụrụ na achọrọ nkwado teknụzụ biko kpọtụrụ ụlọ ọrụ anyị.
5. Jiri mpaghara raara onwe ya nye na pipettes tupu na mgbe nkwalite, na-eyi uwe mgbe ị na-arụ ọrụ ma dochie ha ugboro ugboro;emeghela tube mmeghachi omume mgbe PCR gwụchara iji belata mmetọ nke ihe nlele site na ngwaahịa PCR.
