2×Sensi Direct Premix-UNG (Probe qPCR)
Nomoro ea katse: HCB5151A
SensiDirect Premix-UNG (Probe qPCR) e etselitsoe ho etsa PCR ka kotloloho ho tsoa lisampoleng ntle le ho ntšoa ha DNA kapa ho lokisa sampole.Sehlahisoa sena se na le DNA polymerase e chesang e qalang, uracil DNA glycosylase (UNG), RNase Inhibitor, MgCl.2, dNTPs (ka dUTP sebakeng sa dTTP), le li-stabilizers, bakeng sa palo ea PCR (qPCR).Sehlahisoa sena se emela ho mamellana ho hoholo ha li-inhibitor, 'me ka hona se ka sebelisoa ka kotloloho ho fumants'oa lisampole tse kang 'metso, mathe, mali a anti-coagulated a felletseng, plasma, le serum ntle le ho ntšoa ha DNA.Reagent e sebelisa buffer ea qPCR e nang le li-enzyme tse tsoakiloeng tsa anti-inhibitory DNA polymerase le UNG enzyme.Ka hona, e ka fumana ntlafatso e ntle ea liphatsa tsa lefutso tse shebiloeng ka lisampole tse nang le li-inhibitors le ho thibela matlafatso a fosahetseng a bakoang ke tšilafalo ea PCR le aerosol.Reagent ena e lumellana le lisebelisoa tse ngata tsa PCR tsa fluorescent, tse kang Applied Biosystems, Eppendorf, Bio-Rad, Roche joalo-joalo.
Likaroloana
1. 50×SensiDirect Enzyme/UNG Mix
2. 2×SensiDirect Premix Buffer (dUTP)
Maemo a polokelo
Likarolo tsohle li lokela ho bolokoa ho -20 ℃ bakeng sa polokelo ea nako e telele le 4 ℃ bakeng sa likhoeli tse 3.Ka kopo kopanya hantle ka mor'a ho qhibiliha le centrifuge pele u sebelisa.Qoba ho qhibiliha khafetsa.
Protocol ea Libaesekele
| Mohato | Mocheso | Nako | Baesekele |
| Tšilo ea lijo | 50 ℃ | 2 mets | 1 |
| Polymerase activation | 95℃ | 1-5 mets | 1 |
| Denature | 95℃ | 10-20s | 40-50 |
| Katoloso/ Katoloso | 56-64 ℃ | 20-60s |
Litaelo tsa ho Pipetting
| Reagent | Bolumo ka karabelo | Bolumo ho latela karabelo | Boikutlo ba ho Qetela |
| 2×SensiDirect Premix Buffer (dUTP) | 12.5µL | 25µL | 1× |
| 50×SensiDirect Enzyme/UNG Mix | 0.5µL | 1µL | 1× |
| 25×Primer-Probe Mix1, 2 | 1µL | 2µL | 1× |
| Mohlala3, 4 | - | - | - |
| ddH2O | - | - | - |
| Kakaretso ea molumo | 25 μL | 50 μL | - |
1. Khatiso ea ho qetela ea primer hangata ke 0.2μM.Bakeng sa liphetho tse ntle, mahloriso a primer a ka ntlafatsoa ka har'a mefuta ea 0.2-1μM.
2. Ka kakaretso, mahloriso a probe a ka ntlafatsoa ka har'a sebaka sa 0.1-0.3μM.Khokahano e nepahetseng ea tlhahlobo e amana le sesebelisoa sa matla sa PCR sa nako ea 'nete, mofuta oa probe, le mofuta oa ntho e tšoaeang fluorescent.Ka kopo, sheba bukana ea lisebelisoa kapa litlhoko tse khethehileng tsa probe e 'ngoe le e 'ngoe ea fluorescent.
3. Mefuta e fapaneng ea lisampole e na le mefuta e fapaneng le likahare tsa inhibitor le palo ea kopi ea liphatsa tsa lefutso.Molumo oa mohlala o lokela ho nkoa ka boemo ba sebele.Etsa dilution ea sampole ka ho eketsa metsi a se nang nuclease kapa TE Buffer, ha ho hlokahala.
4. Bolumo bo khothalelitsoeng ba lisampole tse fapaneng:
| Mohlala | Volume bakeng sa 50 e le 'ngoe μL karabelo | Boholo karolelano |
| Anticoagulated mali a feletseng | 2.5 μL | 5% |
| Plasma | 15 μL | 30% |
| Serum | 10 μL | 20% |
| Ho phunya 'metso | 10 μL | 20% |
| Mathe | 10 μL | 20% |
Taolo ea Boleng
1. Ho lemoha ha mosebetsi: kutloisiso, ho khetheha le ho pheta-pheta ha qPCR.
2. Ha ho ts'ebetso ea nuclease exogenous: ha ho exogenous endonuclease le exonuclease tšilafalo.
Lintlha tsa Sehlahisoa
1. Sehlahisoa sena se sebelisa mofuta o mocha oa DNA polymerase e qalang e chesang, e ka sebelisoang ka mor'a metsotso e 1-5. Kaha buffer ea eona ea ho arabela e ntlafalitsoe, e loketse haholoanyane PCR ea palo ea fluorescence e habeli kapa e mengata e sebelisang mokhoa oa ho hlahloba.
2. Haeba boleng ba Rn ba amplification ea PCR bo le tlase haholo kapa ho atolosoa ho hlakile ho thibetsoe, ho fokotsa palo ea sampole, ho eketsa molumo oa karabelo kapa ho hlapolla ha sampula pele ho ka ntlafatsa liphetho.
3. Pokello ea mali, mathe, moroto, 'metso, joalo-joalo e lokela ho latela litlhoko tsa litekanyetso tsa bongaka,' me sampole e ncha e ka sebelisoa ho thibela ho senyeha ha nucleic acid.
4. Kaha li-amplicon tse fapaneng li na le katleho e fapaneng ea tšebeliso ho dUTP le kutloisiso ho UNG, li-reagents li lokela ho ntlafatsoa haeba kutloisiso ea ho lemoha e fokotseha ha u sebelisa tsamaiso ea UNG.Ka kopo ikopanye le rona bakeng sa tšehetso ea tekheniki ha ho hlokahala.
5. Ho qoba ho holisoa ha lihlahisoa tsa PCR lipakeng tsa mehato e le 'ngoe, sebaka sa liteko se inehetseng le pipette lia hlokahala bakeng sa ho holisa.Sebetsa ka liatlana 'me u fetole khafetsa' me u se ke ua bula tube ea karabelo ka mor'a ho holisa PCR.
