Universal SYBR GREEN qPCR Premix (Xiav)
Cat No: HCB5041B
Universal Blue qPCR Master Mix (Dye Based) yog ib qho kev daws teeb meem ua ntej rau 2 × real-time quantitative PCR amplification yam ntxwv los ntawm siab rhiab heev thiab tshwj xeeb, yog xiav xim, thiab muaj cov nyhuv ntawm cov qauv ntxiv tracing.Cov ntsiab lus tseem ceeb ntawm Taq DNA polymerase siv cov tshuaj tiv thaiv kub pib ua haujlwm kom tsis txhob muaj qhov tshwj xeeb amplification vim primer annealing thaum lub sij hawm ua qauv.Nyob rau tib lub sijhawm, cov qauv no ntxiv cov kev txhawb nqa los txhim kho qhov kev ua tau zoo ntawm PCR cov tshuaj tiv thaiv thiab sib npaug ntawm cov noob caj noob ces nrog cov ntsiab lus GC sib txawv (30 ~ 70%), kom ntau PCR tuaj yeem tau txais kev sib raug zoo linear hauv ntau qhov ntau. cheeb tsam.Cov khoom no muaj tshwj xeeb ROX Passive Reference Dye, uas muaj feem xyuam rau feem ntau cov cuab yeej qPCR.Nws tsis yog tsim nyog los kho qhov concentration ntawm ROX ntawm cov twj paj nruag sib txawv.Nws tsuas yog tsim nyog ntxiv primers thiab templates los npaj cov tshuaj tiv thaiv rau kev nthuav dav.
Cheebtsam
Universal Blue qPCR Master Mix
Cia tej yam kev mob
Cov khoom xa tuaj nrog cov pob khov nab kuab thiab tuaj yeem khaws cia ntawm -25 ℃ ~ -15 ℃ rau 18 lub hlis.Nws yog ib qho tsim nyog kom tsis txhob muaj lub teeb pom kev zoo thaum khaws cia lossis npaj cov tshuaj tiv thaiv.
Specification
| Nco ntsoov | 2 × |
| Kev kuaj xyuas txoj kev | SYBR |
| PCR txoj kev | qPCR |
| Polymerase | Cov DNA polymerase |
| Hom qauv | DNA |
| Cov cuab yeej siv | Feem ntau cov cuab yeej qPCR |
| Yam khoom | SYBR premix rau real-time fluorescence quantitative PCR |
| Thov rau (application) | Gene Expression |
Cov lus qhia
1.Reaction System
| Cheebtsam | Qhov siab (μL) | Qhov siab (μL) | Thaum kawg Concentration |
| Universal SYBR GREEN qPCR Premix | 25 | 10 | 1 × |
| Forward Primer (10μmol / L) | 1 | 0.4 | 0.2 μmol / L |
| Rov qab Primer (10μmol / L) | 1 | 0.4 | 0.2 μmol / L |
| DNA | X | X | |
| ddH ua2O | mus 50 | mus 20 | - |
[Ceeb Toom]: Sib tov kom huv si ua ntej siv kom tsis txhob muaj npuas ntau dhau los ntawm kev tuav tes.
a) Primer concentration: Qhov kawg primer concentration yog 0.2μmol / L, thiab tuaj yeem hloov kho ntawm 0.1 thiab 1.0μmol / L raws li qhov tsim nyog.
b) Template concentration: Yog tias lub template yog undiluted cDNA Tshuag tov, lub ntim siv yuav tsum tsis txhob ntau tshaj 1/10 ntawm tag nrho cov ntim ntawm cov tshuaj tiv thaiv qPCR.
c) Template dilution: Nws raug nquahu kom dilute cov tshuaj cDNA Tshuag los ntawm 5-10 zaug.Qhov zoo ntawm cov qauv ntxiv yog qhov zoo dua thaum tus nqi Ct tau los ntawm kev nthuav dav yog 20-30 cycles.
d) Cov tshuaj tiv thaiv kab mob: Nws raug nquahu kom siv 20μL lossis 50μL txhawm rau ua kom muaj txiaj ntsig thiab rov ua dua ntawm lub hom phiaj gene amplification.
e) Kev npaj cov txheej txheem: Thov npaj rau hauv lub rooj zaum huv si thiab siv cov lus qhia thiab cov tshuaj tiv thaiv kab mob uas tsis muaj nuclease residue;nws raug pom zoo kom siv cov lus qhia nrog lim cartridges.Tsis txhob kis kab mob sib kis thiab aerosol paug.
2.Reaction program
Standard Program
| Lub voj voog kauj ruam | Temp. | Sijhawm | Lub voj voog |
| Thawj denaturation | 95 ℃ | 2 min | 1 |
| Denaturation | 95 ℃ | 10 sec | 40 |
| Annealing / Extension | 60 ℃ | 30 sec ★ | |
| Melting nkhaus theem | Instrument Defaults | 1 | |
Txoj Haujlwm ceev
| Lub voj voog kauj ruam | Temp. | Sijhawm | Lub voj voog |
| Thawj denaturation | 95 ℃ | 30 sec | 1 |
| Denaturation | 95 ℃ | 3 sec | 40 |
| Annealing / Extension | 60 ℃ | 20 sec ★ | |
| Melting nkhaus theem | Instrument Defaults | 1 | |
[Faj seeb]: Qhov kev pab cuam ceev yog haum rau feem coob ntawm cov noob, thiab cov txheej txheem kev pab cuam yuav raug sim rau ib tug neeg complex theem nrab cov noob caj noob ces.
a) Annealing kub thiab lub sij hawm: Thov kho raws li qhov ntev ntawm primer thiab lub hom phiaj gene.
b) Fluorescence signal acquisition (★): Thov teev cov txheej txheem sim raws li qhov yuav tsum tau muaj nyob rau hauv cov lus qhia rau kev siv lub ntsuas.
c) Melting nkhaus: Lub ntsuas ntsuas qhov kev pab cuam tuaj yeem siv tau ib txwm.
3. Kev txheeb xyuas qhov tshwm sim
Yam tsawg kawg ntawm peb yam khoom siv roj ntsha tau xav tau rau kev sim ntau.Tom qab cov tshuaj tiv thaiv, lub amplification nkhaus thiab melting nkhaus yuav tsum tau paub tseeb tias.
3.1 Amplification nkhaus:
Tus qauv amplification nkhaus yog S-puab.Kev soj ntsuam ntau yog qhov tseeb tshaj plaws thaum tus nqi Ct poob ntawm 20 thiab 30. Yog tias tus nqi Ct tsawg dua 10, nws yuav tsum tau dilute lub template thiab ua qhov kev sim dua.Thaum tus nqi Ct nyob nruab nrab ntawm 30-35, nws yog ib qho tsim nyog yuav tsum tau ua kom cov template concentration lossis qhov ntim ntawm cov tshuaj tiv thaiv kab mob, txhawm rau txhim kho kev ua kom zoo dua qub thiab ua kom qhov tseeb ntawm cov txiaj ntsig kev soj ntsuam.Thaum tus nqi Ct ntau dua 35, cov txiaj ntsig kev ntsuas tsis tuaj yeem soj ntsuam ntau qhov kev qhia ntawm cov noob, tab sis tuaj yeem siv rau kev soj ntsuam zoo.
3.2 Melting nkhaus:
Lub ncov nkaus xwb ntawm lub melting nkhaus qhia tias cov tshuaj tiv thaiv tshwj xeeb yog qhov zoo thiab kev soj ntsuam kom muaj nuj nqis tuaj yeem ua tau;Yog hais tias lub melting nkhaus qhia tau hais tias ob los yog ntau lub ncov, kev soj ntsuam ntau tsis tuaj yeem ua.Lub melting nkhaus qhia tau hais tias ob lub ncov, thiab nws yog ib qho tsim nyog los txiav txim seb lub ncov uas tsis yog lub hom phiaj yog primer dimer lossis tsis yog qhov tshwj xeeb los ntawm DNA agarose gel electrophoresis.Yog hais tias nws yog primer dimer, nws raug pom zoo kom txo cov primer concentration los yog redesign primers nrog high amplification efficiency.Yog tias nws tsis yog qhov tshwj xeeb amplification, thov nce qhov kub thiab txias, lossis rov tsim cov primers nrog qhov tshwj xeeb.
Sau ntawv
Thov hnav PPE tsim nyog, xws li lub tsho tiv no hauv chav kuaj thiab hnab looj tes, txhawm rau ua kom koj txoj kev noj qab haus huv thiab kev nyab xeeb!
Cov khoom no yog siv rau kev tshawb fawb nkaus xwb!
