Multiplex Imwe Nhanho RT-qPCR Premix-UNG
Katsi Nhamba: HCB5145A
Multiplex Imwe Nhanho RT-qPCR Probe Kit (UDG Plus) ndeye multiplex quantitative PCR kit yakavakirwa paRNA setemplate.Mukuita kwekuedza, reverse transcription uye huwandu hwePCR hwakaitwa muhubhu imwe chete, iyo yakarerutsa kushanda kwekuyedza uye kuderedza njodzi yekusvibiswa.Mune kit iyi, yekutanga strand cDNA yakanyatsogadzirwa nekushisa-inodzivirira Reverse Transcriptase uye yakakwidziridzwa neHotStart Tag DNA Polymerase.Iyo kit inonyanya kuve neyakagadziridzwa MP buffer, enzymes musanganiswa, nezvimwe. Iyo buffer mhinduro yatove neMg.2+uye dNTP.Uye zvakare, izvo zvinhu zvinogona kunyatso kuvharidzira iyo isiri-chaiyo PCR kukwidziridzwa uye kuvandudza mashandiro ekuwedzera kweakawanda qPCR maitiro anowedzerwa, ayo anogona kuve nechokwadi chekusimudzira kushanda uye kuita kusvika kune akawanda amplification reaction.Iyo dUTP/UDG system yakawedzerwa kudzivirira zvinobudirira njodzi yekusvibiswa kweaerosol.
Zvikamu
1. Buffer
2. Enzyme Mix
Tsanangudzo
| Sravana Sameeralu Serial 4th Hot Start | Yakavakwa-mukati inopisa kutanga |
| Nzira yekuona | Primer-probe yekuona |
| PCR nzira | Imwe nhanho RT-qPCR |
| Polymerase | Taq DNA polymerase |
| Rudzi rwemuenzaniso | DNA |
Storage Conditions
Chigadzirwa chacho chinotumirwa nechando chakaoma uye chinogona kuchengetwa pa -25 ~ -15 ℃ kwegore 1.Inofanira kudzivisa kakawandafreeze-thaw.Inokurudzirwa kuchengetedza zvakasiyana.
Mirayiridzo
1.Reaction System
| Zvikamu | Vhoriyamu (μL) | Final Concentration |
| 2 × MP Buffer | 12.5 | 1× |
| Enzyme Mix | 1 | - |
| Primer/Probe musanganiswa (2.5 μM) | 3 | 0.3μM |
| RNA template | 1-10 | - |
| RNase Yemahara H2O | ku25 | - |
Notes:
Iva nechokwadi chekusanganisa zvakanaka usati washandisa, dzivisa mabhuru akawandisa anokonzerwa nekudengenyeka kwechisimba.
a.Primer concentration: Primer musanganiswa unosanganisira multiplex primer, zvichienderana nemamiriro epamusoro primer concentration pamwe pakati pe0.l ne1.0μM.
b.Probe concentration: Probe musanganiswa unosanganisira multiplex probe labeling musiyano wefluorescent boka, zvichienderana nemamiriro epamusoro probe concentration pamwe pakati pe0.05 ne0.5μM.
c.Template dilution: qPCR inonzwa zvakanyanya uye inokurudzirwa kudzikisa template.Kudzora Ct kukosha kwakakodzera pakati pe20 ne35.
d.Kugadzirira kweSistimu: Ndokumbira ugadzirire mune yekupedzisira yakachena tafura yekushanda, uye pipettor uye reaction chubhu isina nuclease yakasara;zvinokurudzirwa kushandisa musoro wepfuti nefilter element.Dzivisa kusvibiswa kwemuchinjikwa uye kusvibiswa kweaerosol.
2. Optimized CyclingProtocol
| Cycle step | Temp. | Nguva | Cycles |
| Reverse transcription | 50 ℃a | 20mins | 1 |
| Kutanga-denaturation | 95℃ | 5 mins | 1 |
| Amplification reaction | 95℃ | 15sec |
40-45 |
| 60 ℃ b | 30sec c |
Notes:
a.Reverse transcription: Tembiricha inogona kusarudza 42°C kana 50°C.
b.Amplification reaction: Iyo tembiricha inogadziriswa zvinoenderana neiyo Tm kukosha kweiyo yakagadzirirwa primers.
c.Fluorescence chiratidzo chekutora: Ndokumbira isa maitiro ekuyedza zvinoenderana nezvinodiwa zvechiridzwa chekushandisa.
Notes
Ndokumbira upfeke iyo PPE inodiwa, senge lab jasi uye magurovhosi, kuve nechokwadi chehutano hwako uye kuchengetedzeka.
