5 × Neoscript Fast RT-qPCR Premix plus-UNG
Katsi Nhamba: HCB5142A
Neoscript Fast RT Premix-UNG (Probe qRT-PCR) yakagadzikana zvakanyanya imwe-chubhu probe-based musanganiswa wakakodzera kune imwe-nhanho reverse transcription uye uwandu PCR (qRT-PCR).Inotsigira pre-kusanganiswa kweprimers uye probes uye inogara yakagadzikana mushure mekuchengetedza kwenguva yakareba pane yakaderera tembiricha.Sample inoedzwa inogona kuwedzerwa zvakananga kana uchishandisa, pasina yekuwedzera chubhu kuvhura / pombi kushanda.Ichi chigadzirwa chinopa zvikamu, semuenzaniso kupisa-kutanga DNA polymerase, M-MLV, kupisa-labile uracil DNA glycosylase (TS-UNG), RNase Inhibitor, MgCl.2, dNTPs (ine dUTP pachinzvimbo che dTTP), uye zvinodzikamisa.Neiyo genetically modified yekukurumidza amplification reverse transcriptase uye DNA polymerase, zvinokwanisika kupedzisa PCR amplification mukati memaminitsi makumi maviri kusvika makumi mana.Iyi reagent inoshandisa yakakosha buffer yeqPCR ine akasanganiswa enzymes eanti-inhibitory amplification enzyme uye UNG enzyme.Naizvozvo, inogona kuwana kukwidziridzwa kwakanaka kwemajini ekunangwa uye kudzivirira kukwidziridzwa kwenhema kunokonzerwa nePCR yakasara uye kusvibiswa kweaerosol.Ichi reagent inoenderana neakawanda fluorescence quantitative PCR zviridzwa kubva kuvagadziri vakaita seApplied Biosystems, Eppendorf, Bio-Rad uye Roche.
Chikamu
1.25×Neoscript Fast RTase/UNG Mix
2.5 × Neoscript Fast RT Premix Buffer (dUTP)
Storage Conditions
Zvese zvikamu zvinofanirwa kuchengetwa pa -20 ℃ yekuchengetedza kwenguva refu uye 4 ℃ kusvika kumwedzi mitatu.Ndokumbira unyatso sanganisa mushure mekunyunguduka uye centrifuge usati washandisa.Dzivisa kugaroomesa nechando.
qRT-PCR Reaction System Kugadzirira
| Zvikamu | 25μLSystem | 50μLSystem | Final Concentration |
| 5 × Neoscript Fast RT Premix Buffer (dUTP) | 5μL | 10μL | 1× |
| 25×Neoscript Fast RTase/UNG Mix | 1μL | 2μL | 1× |
| 25 × Primer-Probe Mixa | 1μL | 2μL | 1× |
| RNA templateb | - | - | - |
| ddH2O | Kusvika ku25μL | Kusvika ku50μL | - |
1) a.Mamiriro ekupedzisira ekutanga anowanzo 0.2μM.Kuti uwane mhedzisiro iri nani, iyo primer concentration inogona kuvandudzwa mukati meiyo 0.2-1μM.Kazhinji, iyo probe concentration inogona kuvandudzwa mukati meiyo 0.1-0.3μM.
2) b.Paunenge uchishandisa kukurumidza PCR Procedure, kuwedzera kuwanda kweprimers uye probes kunogona kukonzera zvirinani amplification mhedzisiro, uye chiyero chavo chinofanira kugadziridzwa zvinoenderana.
3) Mhando dzakasiyana dzemasampuli dzine mhando dzakasiyana uye zvirimo zveinhibitor uye kopi nhamba yechinangwa gene.Muenzaniso wevhoriyamu unofanirwa kutariswa nemamiriro chaiwo.Ita dilution yemuenzaniso nemvura isina nuclease kana TE Buffer, kana zvichidikanwa.
Reaction Conditions
| Nguva dzose PCR Maitiro | Fast PCR Maitiro | ||||||
| Maitiro | Temp. | Nguva | Cycle | Maitiro | Temp. | Nguva | Cycle |
| Reverse Transcription | 50 ℃ | 10-20mins | 1 | Reverse Transcription | 50 ℃ | 5 mins | 1 |
| Polymerase Activation | 95℃ | 1-5 mins | 1 | Polymerase Activation | 95℃ | 30s | 1 |
| Denaturation | 95℃ | 10-20s | 40-50 | Denaturation | 95℃ | 1-3s | 40-50 |
| Annealing uye Extension | 56-64 ℃ | 20-60s | Annealing uye Extension | 56-64 ℃ | 3-20s | ||
Quality Control
1.Basa rekuona: kunzwa, kujeka uye kudzokorora kweqPCR.
2.Hapana exogenous nuclease chiitiko: hapana exogenous endonuclease uye exonuclease kusvibiswa.
Notes
1.Amplification rate yekukurumidza DNA polymerase haisi pasi pe1kb/10s.Zvishandiso zvakasiyana zvePCR zvine kumhanya kwakasiyana kwekudziya uye kutonhora, maitiro ekudzora tembiricha uye kupisa kwekupisa, uye nekudaro optimization yeprimer yako / probe yekutarisa uye yekumhanya nzira musanganiswa neyako yekukurumidza PCR chiridzwa kwakakosha.
2.Ichi chigadzirwa chinoita kushanda kwakakura, uye chakakodzera kune yakakwira-sensitive molecular diagnosis.Matanho matatu PCR nzira inokurudzirwa kune maprimers ane yakaderera annealing tembiricha kana yekukudza zvimedu zvirefu kupfuura 200 bp.
3.Sezvo maamplicon akasiyana ane akasiyana ekushandisa kwe dUTP uye akasiyana sensitivity kune UNG, ma reagents anofanirwa kuvandudzwa kana iyo yekuona senitivity yakadzikira kana uchishandisa UNG system.Ndapota taura nesu kuti tiwane rubatsiro rwemhizha kana zvichidiwa.
4.Kuti udzivise kukwidziridzwa kwezvigadzirwa zvePCR, nzvimbo yakatsaurirwa yekuyedza uye pipette inodiwa pakukwidziridzwa.Shanda nemagurovhosi uye shandura kazhinji uye usavhure PCR chubhu mushure mekukwidziridzwa.
