Multiplex qPCR Probe Premix
Katsi Nhamba: HCB5051A
TaqMan multiplex qPCR Master Mix (Dye Based) ndeye pre-mhinduro ye 2 × chaiyo-nguva yakawanda PCR amplification inoratidzwa nehupamhi hwekunzwa uye hunhu, iyo yebhuruu muruvara, uye ine mhedzisiro yekuwedzera sampuli.Ichi chigadzirwa ndeye 2 × Musanganiswa pre-yakasanganiswa reagent inogonesa inosvika ina fluorescent quantitative PCR maitiro mune imwe chete maitiro zvakanaka.Ichi chigadzirwa chine genetically modified antibody nzira yekupisa-kutanga Taq enzyme, inovandudza zvakanyanya kunzwisiswa uye hunhu.Panguva imwecheteyo, chigadzirwa ichi chakanyatsogadzirisa iyo yakawanda-reaction buffer, iyo inogona kuvandudza kugona kwekuita kwekuita uye kukurudzira kukwidziridzwa kunoshanda kweyakaderera-concentration templates.Ichi chigadzirwa chinogona kushandiswa genotyping uye multiplex quantitative ongororo.
Tsanangudzo
| Sravana Sameeralu Serial 4th Hot Start | Yakavakwa-mukati inopisa kutanga |
| Nzira yekuona | Primer-probe yekuona |
| PCR nzira | qPCR |
| Polymerase | Taq DNA polymerase |
| Rudzi rwemuenzaniso | DNA |
Storage Conditions
Chigadzirwa chacho chinotumirwa nechando chakaoma uye chinogona kuchengetwa pa -25 ~ -15 ℃ kwe2 gore.
Mirayiridzo
1. KuitaSystem
| Zvikamu | Vhoriyamu (μL) | Final Concentration |
| 2 × TaqMan multiplex qPCR Master Mix | 12.5 | 1× |
| Musanganiswa wekutanga (10 μmol/L) a | × | 0.1 - 0.5 μmol/L |
| Probe musanganiswa (10 μmol/L)b | × | 50 - 250 nmol/L |
| Rox reference dhayi | 0.5 | 1× |
| Template DNA/cDNA | 1-10 | - |
| ddH2O | kusvika ku25 | - |
Notes:Sanganisa zvakakwana usati washandisa kudzivirira mabhuru akawandisa kubva pakuzunungusa kwakasimba.
a.Primer concentration: Primer Mix ine mapeya akawanda emaprimers, kazhinji yekutanga imwe neimwe pachikamu chekupedzisira che 0.2 μmol / L uye inogonawo kugadziriswa pakati pe 0.1 uye 0.5 μmol / L sezvakakodzera.
b.Probe concentration: Probe Mix ine akawanda maprobe ane akasiyana fluorescence masiginecha, uye kusungirirwa kwechero probe kunogona kugadziriswa pakati pe50 ne250 nmol/L zvinoenderana nemamiriro chaiwo.
1.Rox dhayi referensi:Inoshandiswa paChaiyo Nguva PCR yekusimudzira chiridzwa seApplied Biosystems kugadzirisa kukanganisa kweiyo fluorescence chiratidzo chinogadzirwa pakati pematsime;chigadzirwa ichi hachina Rox dhayi referensi.Cas#10200 inokurudzirwa kana ichidikanwa.
2.Template dilution: qPCR ine hunyoro hwakanyanya, uye inokurudzirwa kudzikisa template kuti ishandiswe.Kana iyo template iri cDNA stock solution, iyo template volume haifanire kudarika 1/10 yehuwandu hwehuwandu.
3.Reaction system: 25μL, 30μL kana 50 μL inokurudzirwa kuti ive nechokwadi chekushanda uye kudzokorora kwechinangwa chekusimudzira gene.
4.Kugadzirira kweSistimu: Ndokumbira ugadzirire mubhenji rakachena kwazvo, uye shandisa iwo matipi uye maitiro machubhu pasina masara nuclease;zvinokurudzirwa kushandisa mazano ane filter cartridges.Dzivisa kusvibiswa kwemuchinjikwa uye kusvibiswa kweaerosol.
2.Reaction program
| Cycle step | Temp. | Nguva | Cycles |
| Kutanga-denaturation | 95 ℃ | 5 mins | 1 |
| Denaturation | 95 ℃ | 15sec | 45 |
| Kuwedzera/Kuwedzera | 60 ℃ | 30sec |
Notes:
1.Annealing / Extension: Tembiricha uye nguva inogona kugadziridzwa zvakakodzera zvinoenderana neyakagadzirirwa primer Tm kukosha.
2.Fluorescence chiratidzo chekutora: Iyo fluorescence chiratidzo chekutora nguva inodiwa kune akasiyana qPCR yekuona zviridzwa zvakasiyana, ndapota isa zvinoenderana nediki nguva yakatarwa.Nguva yezviridzwa zvakawanda zvakajairika inoiswa sezvizvi:
20 sec: Applied Biosystems 7700, 7900HT, 7500 Fast
31 sec: Yakashandiswa Biosystems 7300
32 sec: Inoshandiswa Biosystems 7500
Notes
Ndokumbira upfeke PPE inodiwa, jasi rakadaro uye magurovhosi, kuve nechokwadi chehutano hwako uye kuchengetedzeka!
