Mohato o le mong RT-qPCR SYBR Green Premix
Nomoro ea katse: HCB5140A
Mohato o le Mong RT-qPCR Syber Green Premix ke ea fluorescence quantification e thehiloeng ho SYBR Green I dae.Ho sebelisoa li-primers tse ikhethileng tsa lefutso, reverse transcription le qPCR reactions li phethoa ka thapo e le 'ngoe, ho felisa tlhoko ea ho buloa khafetsa le ts'ebetso ea lipeipi, ho ntlafatsa haholo ts'ebetso ea tlhahlobo le ho fokotsa kotsi ea tšoaetso.Bakeng sa disampole tsa RNA, kit e sebelisa Reverse Transcriptase e thibelang mocheso bakeng sa motsoako o sebetsang hantle oa cDNA le HotStart Taq DNA Polymerase bakeng sa matlafatso ea bongata.Tlas'a sisteme e ntlafalitsoeng ea buffer, kutlo ea kit e ka ba holimo joalo ka 0.1 pg bakeng sa lipheo tse hlahisitsoeng haholo le holimo ho 1 pg bakeng sa lipheo tse boletsoeng ka mokhoa o itekanetseng.Kit e loketse ho holisa le ho lekanya lisampole tsa DNA.E thusa ho lemoha le ho lekanya li-nucleic acid ho tsoa lisampole tse fapaneng tsa limela le liphoofolo, lisele le likokoana-hloko.
Likaroloana
| No | Lebitso | Bolumo | Bolumo |
| 1 | Buffer e tsoetseng pele | 250 μL | 2×1.25 mL |
| 2 | Motsoako o tsoetseng pele oa enzyme | 20 μL | 200 μL |
| 3 | RNase Free H2O | 250 μL | 2×1.25 mL |
Maemo a polokelo
Sehlahisoa sena se lokela ho bolokoa ho -25 ~ -15 ℃ hole le leseli bakeng sa selemo se le seng.
Litaelo
1.Reaction system tlhophisod
| Likaroloana | Bolumo (μL) | Bolumo (μL) | Khatiso ea ho qetela |
| Buffer e tsoetseng pele | 12.5 | 25 | 1× |
| Motsoako o tsoetseng pele oa enzyme | 1 | 2 | - |
| Primer ea pele (10 μmol/L)a | 0.5 | 1 | 0.2 μmol/L |
| Primer e khutlisang (10 μmol/L)a | 0.5 | 1 | 0.2 μmol/L |
| Setšoantšo sa RNAb | X | X | - |
| RNase Free H2Oc | ho ea ho 25 | ho ea ho 50 | - |
Lintlha:
1) a.Tmahloriso a hae a ho qetela e ne e le 0.2 μmol/L, e neng e ka boela ea fetoloa pakeng tsa 0.1 le 1μmol/L ha ho loketse.
2) b.Reagent e na le kutloelo-bohloko ka ho fetisisa, e nang le Total RNA ka har'a 1pg-1μg, 'me tlhahlobo ea lisampole tsa batho e bontšitse tlhahiso e nepahetseng ea 1 pg-100 ng, e laolang boleng ba Ct ka kakaretso ho 15-30 ha ho loketse.
3) c.Ho khothaletsoa ho sebelisa 20μL kapa 50μL ho netefatsa bonnete le ho ikatisa ha liphatsa tsa lefutso tsa sepheo.
4) d.Ka kopo itokisetse bencheng e hloekileng haholo 'me u sebelise malebela a se nang masala a nuclease le li-tubes tsa reaction;malebela a nang le li-cartridge tsa filthara lia khothaletsoa.Qoba tšilafalo e fapaneng le tšilafalo ea aerosol.
2.Lenaneo la ho arabela
| Mohato oa potoloho | Temp. | Nako | Lipotoloho |
| Phetolelo ea morao | 50 ℃a | 6 mets | 1 |
| Denaturation ea pele | 95℃ | 5 mets | 1 |
| Boitšoaro ba amplification | 95℃ | 15 metsotsoana | 40 |
| 60 ℃b | 30 metsotsoana | ||
| Mokhahlelo oa ho qhibiliha | Mehato ea Liletsa | 1 | |
Lintlha:
1) a.Mocheso o ka morao oa khatiso o ka khethoa pakeng tsa 50-55 ° C ho latela litlhoko tsa liteko.Bakeng sa disampole tsa DNA, mokhoa oa ho fetolela o ka morao o ka siuoa.
2) b.Maemong a khethehileng mocheso oa annealing / extension o ka fetoloa ho ea ka boleng ba pele ba Tm, 60 ° C e buelloa.
Lintlha
1. Sehlahisoa sena ke sa tšebeliso ea lipatlisiso feela.
2. Ka kopo sebetsa ka lijase tsa lab le liatlana tse lahloang, molemong oa polokeho ea hau.
