2×Sensi Direct Premix-UNG (Probe qPCR)
Katsi Nhamba: HCB5151A
SensiDirect Premix-UNG (Probe qPCR) yakagadzirirwa kuita PCR zvakananga kubva kumasampuli pasina kutorwa kweDNA kana kugadzirira sampuli.Iyi reagent ine inopisa-kutanga DNA polymerase, uracil DNA glycosylase (UNG), RNase Inhibitor, MgCl.2, dNTPs (ine dUTP pachinzvimbo che dTTP), uye zvinodzikamisa, zvehuwandu PCR (qPCR).Iyi reagent inofanotungamira yakakwira inhibitor-kushivirira, uye nekudaro inogona kuiswa zvakananga pakuonekwa kwemasamples senge pahuro swab, mate, anti-coagulated ropa rese, plasma, uye serum isina kubviswa kweDNA.Iyo reagent inoshandisa proprietary buffer yeqPCR ine yakasanganiswa enzymes ye-anti-inhibitory DNA polymerase uye UNG enzyme.Naizvozvo, inogona kuwana kukwidziridzwa kwakanaka kwemajini ekunangwa mumasampuri ane inhibitors uye inhibit yenhema yakanaka amplification inokonzerwa nePCR yasara uye aerosol kusvibiswa.Iyi reagent inoenderana neakawanda fluorescent quantitative PCR zviridzwa, seApplied Biosystems, Eppendorf, Bio-Rad, Roche zvichingodaro.
Zvikamu
1. 50×SensiDirect Enzyme/UNG Mix
2. 2×SensiDirect Premix Buffer (dUTP)
Kuchengetedza mamiriro
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.
Cycling Protocol
| Danho | Tembiricha | Nguva | Cycle |
| Kugaya | 50 ℃ | 2min | 1 |
| Polymerase activation | 95℃ | 1-5 min | 1 |
| Denature | 95℃ | 10-20s | 40-50 |
| Kuwedzera/Kuwedzera | 56-64 ℃ | 20-60s |
Pipetting Instructions
| Reagent | Volume per reaction | Volume per reaction | Final Concentration |
| 2×SensiDirect Premix Buffer (dUTP) | 12.5µL | 25µL | 1× |
| 50×SensiDirect Enzyme/UNG Mix | 0.5µL | 1µL | 1× |
| 25 × Primer-Probe Mix1, 2 | 1µL | 2µL | 1× |
| Muenzaniso3, 4 | - | - | - |
| ddH2O | - | - | - |
| Vhoriyamu yese | 25 μL | 50 μL | - |
1. Mhedziso yekupedzisira yeprimer inowanzoita 0.2μM.Kuti uwane mhedzisiro iri nani, iyo primer concentration inogona kuvandudzwa mukati meiyo 0.2-1μM.
2. Kazhinji, kuongororwa kweprobe kunogona kugadziriswa mukati mehuwandu hwe 0.1-0.3μM.Iyo yakakwana yekumisikidzwa kweiyo probe ine hukama neiyo chaiyo nguva PCR amplification chiridzwa, rudzi rwekuferefeta, uye rudzi rwefluorescent labeling chinhu.Ndokumbira utarise kune chinyorwa chezviridzwa kana izvo zvinodiwa zvega yega fluorescent probe.
3. Mhando dzakasiyana dzemasampuli dzine marudzi akasiyana-siyana uye zvinyorwa zve inhibitor uye nhamba yekopi yejini rinotarirwa.Muenzaniso wevhoriyamu unofanirwa kutariswa nemamiriro chaiwo.Ita dilution yemuenzaniso nekuwedzera nuclease-isina mvura kana TE Buffer, kana zvichidikanwa.
4. Yakakurudzirwa vhoriyamu yemasampuli akasiyana:
| Muenzaniso | Vhoriyamu kune imwe 50 μL reaction | Maximum ratio |
| Anticoagulated ropa rose | 2.5 μL | 5% |
| Plasma | 15 μL | 30% |
| Serum | 10 μL | 20% |
| Huro swab | 10 μL | 20% |
| Mate | 10 μL | 20% |
Quality Control
1. Kuonekwa kwebasa: kunzwa, kujeka uye kudzokorora kweqPCR.
2. Hapana exogenous nuclease basa: hapana exogenous endonuclease uye exonuclease kusvibiswa.
Zvinyorwa zveChigadzirwa
1. Ichi chigadzirwa chinoshandisa chinyorwa chemhando inopisa-kutanga DNA polymerase, iyo inogona kushandiswa mumaminitsi 1-5.Sezvo maitiro ayo buffer akagadziridzwa, inonyanya kukodzera kaviri kana yakawanda fluorescence quantitative PCR uchishandisa nzira yekuongorora.
2. Kana iyo Rn kukosha kwePCR amplification yakanyanya kuderera kana kuwedzera kuri pachena kwakadziviswa, kuderedza chiyero chemuenzaniso, kuwedzera reaction volume kana yapfuura dilution yemuenzaniso inogona kuvandudza migumisiro.
3. Kuunganidzwa kweropa, mate, weti, swab yehuro, zvichingodaro kunofanirwa kutevedzera zvinodiwa nekiriniki, uye sampuli nyowani inogona kushandiswa kudzivirira kushatisa nucleic acid.
4. Sezvo maamplicon akasiyana ane kushandiswa kwakasiyana kwekushandisa kune dUTP uye kunzwisisika kune UNG, ma reagents anofanirwa kugadziriswa kana kunzwisiswa kwekuona kunoderera pakushandisa UNG system.Ndapota taura nesu kuti tiwane rubatsiro rwemhizha kana zvichidiwa.
5. Kuti udzivise kukwidziridzwa kwezvigadzirwa zvePCR pakati pekuita nhanho imwe, nzvimbo yakatsaurirwa yekuyedza uye pipette inodiwa kuti iwedzere.Shanda nemagurovhosi uye shandura kazhinji uye usavhure reaction chubhu mushure mekukwidziridzwa kwePCR.
