5× Neoscript Degdeg ah RT-qPCR Premix iyo UNG
Bisadda No: HCB5142A
Neoscript Fast RT Premix-UNG (Probe qRT-PCR) waa isku-dhafan hal-tubo ah oo ku-saleysan baaritaan aad u deggan oo ku habboon hal-tallaabo qoraal-ku-noqosho iyo PCR tirada (qRT-PCR).Waxay taageertaa isku-darka hore ee asaasiga ah iyo shaybaarrada waxayna joogtaa xasilloonida wakhti dheer ka dib kaydinta heerkulka hooseeya.Muunadda la tijaabinayo si toos ah ayaa loogu dari karaa marka la isticmaalayo, iyada oo aan la helin hawl-gelin tuubo oo dheeraad ah.Alaabtani waxay bixisaa qaybo, tusaale kuleyl-bilawga DNA polymerase, M-MLV, kulayl-labile uracil DNA glycosylase (TS-UNG), RNase Inhibitor, MgCl2, dNTPs (oo leh dUTP halkii dTTP), iyo xasiliyayaal.Iyada oo la adeegsanayo hiddo-weyneysiinta degdegga ah ee beddelka transcriptase iyo DNA polymerase, waxaa suurtogal ah in lagu dhammaystiro kordhinta PCR 20-40 daqiiqo gudahood.Reagent-kani waxa uu u isticmaala kayd gaar ah qPCR oo leh ensaymes isku dhafan oo ah enzym xoojinta ka-hortagga ah iyo enzyme UNG.Sidaa darteed, waxay heli kartaa kordhinta wanaagsan ee hiddo-wadaha bartilmaameedka ah waxayna ka hortagtaa kordhinta beenta ah ee ay keento wasakhowga PCR iyo hawada hawada.Reagent-kani waxa uu la jaanqaadi karaa inta badan qalabka PCR ee tirooyinka fluorescence ee ka yimaadda soosaarayaasha sida Applied Biosystems, Eppendorf, Bio-Rad iyo Roche.
Qayb
1.25× Neoscript Fast RTase/UNG Mix
2.5× Neoscript Degdeg ah RT Premix Buffer (dUTP)
Xaaladaha Kaydinta
Dhammaan qaybaha waa in lagu hayaa -20 ℃ kaydinta muddada-dheer iyo 4℃ ilaa 3 bilood.Fadlan si fiican isku qas dhalaalinta ka dib oo centrifuge ka hor inta aanad isticmaalin.Iska ilaali baraf-dhalaalid oo joogto ah.
qRT-PCR Diyaarinta Nidaamka Falcelinta
Qaybaha | 25μLNidaamka | 50μLNidaamka | U-fiirsashada kama dambaysta ah |
5× Neoscript Degdeg ah RT Premix Buffer (dUTP) | 5μL | 10μL | 1× |
25× Neoscript Fast RTase/UNG Mix | 1μL | 2μL | 1× |
25×Primer-Probe Mixa | 1μL | 2μL | 1× |
Template RNAb | - | - | - |
ddH2O | Ilaa 25μL | Ilaa 50μL | - |
1) a.U fiirsiga ugu dambeeya ee asaasiga ah badanaa waa 0.2μM.Si loo helo natiijooyin ka wanaagsan, xoogga saarista asaasiga ah waxaa lagu hagaajin karaa inta u dhaxaysa 0.2-1μM.Guud ahaan, xoogga saarista baaritaanka waxaa lagu hagaajin karaa inta u dhexeysa 0.1-0.3μM.
2) b. Markaad isticmaasho PCR-da degdega ah, kordhinta xoogga saarida asaasiga ah iyo baarayaasha waxay keeni kartaa natiijooyin wax-weyneyn oo wanaagsan, saamigoodana waa in si habboon loo hagaajiyaa.
3) Noocyada kala duwan ee muunado waxay ka kooban yihiin noocyo kala duwan iyo nuxurka horjoogayaasha iyo koobiga tirada hidda-wadaha.Mugga muunada waa in lagu tixgaliyaa xaalad dhab ah.Ku qas muunadda muunadda biyo-la'aanta nukliyeerka ama TE Buffer, haddii loo baahdo.
Falcelinta Cshuruudaha
Habka PCR ee joogtada ah | Habka PCR degdega ah | ||||||
Habraaca | Heerkulka | Waqtiga | Wareegto | Habraaca | Heerkulka | Waqtiga | Wareegto |
Dib u qorista | 50℃ | 10-20 daqiiqo | 1 | Dib u qorista | 50℃ | 5 daqiiqo | 1 |
Polymerase Firfircoonida | 95℃ | 1-5 daqiiqo | 1 | Polymerase Firfircoonida | 95℃ | 30s | 1 |
Denaturation | 95℃ | 10-20s | 40-50 | Denaturation | 95℃ | 1-3s | 40-50 |
Annealing iyo Kordhinta | 56-64℃ | 20-60s | Annealing iyo Kordhinta | 56-64℃ | 3-20s |
Xakamaynta tayada
1.Ogaanshaha shaqada: dareenka, gaar ahaaneed iyo ku celcelinta qPCR.
2.Wax dhaqdhaqaaq nuclease ah ma jiro: ma jiro endonuclease exogenous iyo wasakhowga exonuklease.
Xusuusin
1.Heerka kordhinta polymerase DNA ee degdega ah kama yarayn 1kb/10s.Aaladaha PCR ee kala duwan waxay leeyihiin xawli kuleyl iyo qaboojin oo kala duwan, hababka xakamaynta heerkulka iyo dhaqdhaqaaqa kulaylka, sidaas awgeed kor u qaadida feejignaantaada asaasiga ah iyo habka socodsiinta marka lagu daro qalabkaaga PCR ee degdega ah waa lama huraan.
2.Alaabtani waxay qabataa dabaqitaan ballaaran, waxayna ku habboon tahay ogaanshaha molecular-ka ee xasaasiga ah.Habka PCR ee saddex-tallaabo ah ayaa lagula talinayaa in loo isticmaalo heerkulbeeg hooseeya ama si loo kordhiyo jajabyada dhaadheer ee ka sarreeya 200 bp.
3.Maadaama amplicons kala duwan ay leeyihiin waxtarka isticmaalka dUTP kala duwan iyo dareenka kala duwan ee UNG, reagents waa in la hagaajiyaa haddii dareenka ogaanshaha hoos u dhaco marka la isticmaalayo nidaamka UNG.Fadlan nala soo xidhiidh taageero farsamo haddii loo baahdo.
4.Si looga fogaado in la kordhiyo alaabada PCR, aag tijaabo ah oo go'an iyo pipette ayaa loo baahan yahay si loo kordhiyo.Ku shaqee galoofyada oo si joogta ah u beddel oo ha furin tuubada PCR ka dib marka la kordhiyo.