Multiplex qPCR Probe Premix
Nomoro ea katse: HCB5051A
TaqMan multiplex qPCR Master Mix (Dye Based) ke tharollo ea pele bakeng sa 2 × real-time quantitative quantitative PCR e khetholloang ka kutloisiso e phahameng le ho khetheha, e nang le 'mala o moputsoa, 'me e na le phello ea ho kenyelletsa sampole.Sehlahisoa sena ke 2× Mix pre-mixed reagent e nolofalletsang ho fihla ho tse 'ne karabelo ea fluorescent quantitative PCR ka karabelo e le 'ngoe hantle.Sehlahisoa sena se na le mokhoa oa antibody o fetotsoeng ka liphatsa tsa lefutso ho qala enzyme ea Taq, e ntlafatsa haholo kutlo le ho hlaka.Ka nako e ts'oanang, sehlahisoa sena se ntlafalitse haholo "multi-reaction buffer", e leng se ka ntlafatsang katleho ea karabelo le ho holisa ntlafatso e sebetsang ea litempele tse sa tebang haholo.Sehlahisoa sena se ka sebelisoa ho hlahloba genotyping le multiplex quantitative analysis.
Tlhaloso
| Hot Qala | Ho qala ho chesa |
| Mokhoa oa ho lemoha | Ho fumanoa ka lekhetlo la pele |
| Mokhoa oa ho sebelisa PCR | qPCR |
| Polymerase | Taq DNA polymerase |
| Mofuta oa mohlala | DNA |
Maemo a polokelo
Sehlahisoa se romelloa ka leqhoa le omileng 'me se ka bolokoa ho -25 ~ -15 ℃ bakeng sa selemo sa 2.
Litaelo
1. BoitšoaroTsamaiso
| Likaroloana | Bolumo (μL) | Boikutlo ba ho Qetela |
| 2 × TaqMan multiplex qPCR Master Mix | 12.5 | 1× |
| Motsoako oa pele (10 μmol/L) a | × | 0.1 - 0.5 μmol/L |
| Motsoako oa probe (10 μmol/L)b | × | 50 - 250 nmol/L |
| Dae ea referense ea Rox | 0.5 | 1× |
| Setšoantšo sa DNA/cDNA | 1-10 | - |
| ddH2O | ho fihlela ho 25 | - |
Lintlha:Kopanya hantle pele o sebelisoa ho qoba li-bubble tse feteletseng ho sisinyeha ho matla.
a.Primer concentration: Primer Mix e na le lipara tse ngata tsa li-primers, hangata primer e 'ngoe le e' ngoe sebakeng sa ho qetela sa 0.2 μmol / L 'me e ka boela ea fetoloa pakeng tsa 0.1 le 0.5 μmol / L ha ho loketse.
b.Probe concentration: Probe Mix e na le li-probe tse ngata tse nang le matšoao a fapaneng a fluorescence, 'me mahloriso a probe ka 'ngoe a ka lokisoa lipakeng tsa 50 le 250 nmol/L ho latela maemo a itseng.
1.Referense ea dae ea Rox:E sebelisoa ho sesebelisoa sa amplification sa Real Time PCR joalo ka Applied Biosystems ho lokisa phoso ea lets'oao la fluorescence le hlahisitsoeng lipakeng tsa liliba;sehlahisoa sena ha se na Rox dae reference.Cas#10200 e khothaletsoa ha ho hlokahala.
2.Template dilution: qPCR e hlokolosi haholo, 'me e khothaletsoa ho hlapolla thempleite hore e sebelisoe.Haeba template e le cDNA stock solution, volume ea template ha ea lokela ho feta 1/10 ea kakaretso ea kakaretso.
3.Sistimi ea karabelo: 25μL, 30μL kapa 50 μL e khothaletsoa ho netefatsa katleho le ho pheta-pheta ha tokiso ea liphatsa tsa lefutso.
4.Tokiso ea sistimi: Ka kopo itokisetse bencheng e hloekileng haholo, 'me u sebelise malebela le li-tubes tsa karabelo ntle le masala a nuclease;ho kgothaletswa ho sebelisa malebela ka li-cartridges tsa filter.Qoba tšilafalo e fapaneng le tšilafalo ea aerosol.
2.Lenaneo la ho arabela
| Mohato oa potoloho | Temp. | Nako | Lipotoloho |
| Tšimoloho-ea pele | 95 ℃ | 5 mets | 1 |
| Denaturation | 95 ℃ | 15sec | 45 |
| Katoloso/ Katoloso | 60 ℃ | 30sec |
Lintlha:
1.Annealing / Extension: Thempereichara le nako li ka lokisoa ka nepo ho latela boleng bo hlophisitsoeng ba Tm.
2.Phano ea lets'oao la Fluorescence: Nako ea ho fumana lets'oao la fluorescence e hlokehang bakeng sa lisebelisoa tse fapaneng tsa ho lemoha qPCR e fapane, ka kopo beha ho latela moeli o fokolang oa nako.Nako ea lisebelisoa tse 'maloa tse tloaelehileng e behiloe ka tsela e latelang:
20 sec: Applied Biosystems 7700, 7900HT, 7500 Fast
31 sec: Applied Biosystems 7300
32 sec: Applied Biosystems 7500
Lintlha
Ka kopo, roala PPE e hlokahalang, jase e joalo ea lab le liatlana, ho netefatsa bophelo bo botle le polokeho ea hau!
