Multiplex qPCR Probe Premix
Inombolo yekati: HCB5051A
I-TaqMan multiplex qPCR Master Mix (i-Dye Based) sisisombululo sangaphambili se-2 × i-real-time quantitative quantitative PCR ebonakaliswe ngovakalelo oluphezulu kunye neenkcukacha, eziluhlaza okwesibhakabhaka ngombala, kwaye zinefuthe lokongezwa kwesampulu.Le mveliso yi-2 × Umxube odityaniswe ngaphambili wereagent eyenza ukuya kuthi ga kwi-fluorescent quantitative reactions ye-PCR kwi-reaction enye kakuhle.Le mveliso iqulethe indlela ye-antibody eguqulwe ngokwemfuza yokutshisa i-Taq enzyme, iphucula kakhulu uvakalelo lokukhulisa kunye neenkcukacha.Kwangaxeshanye, le mveliso iye yaphucula ngokunzulu i-buffer ye-reaction multi-reaction, enokuphucula ukusebenza kakuhle kwe-amplification ye-reaction kunye nokukhuthaza ukukhulisa okusebenzayo kweetemplates eziphantsi.Le mveliso ingasetyenziselwa uhlalutyo lwe-genotyping kunye ne-multiplex yobuninzi.
Inkcazo
| Isiqalo esishushu | Isiqalo esishushu esakhelwe ngaphakathi |
| Indlela yokubona | Ukufunyanwa kwe-Primer-probe |
| Indlela yePCR | qPCR |
| Ipolymerase | Taq DNA polymerase |
| Uhlobo lwesampulu | DNA |
Iimeko zokuGcina
Imveliso ithunyelwa ngeqhwa elomileyo kwaye ingagcinwa kwi -25 ~ -15 ℃ iminyaka eyi-2.
Imiyalelo
1. UkusabelaInkqubo
| Amacandelo | Umthamo (μL) | Ukugxininiswa kokugqibela |
| 2× TaqMan multiplex qPCR Master Mix | 12.5 | 1× |
| Umxube wePrimer (10 μmol/L) a | × | 0.1 - 0.5 μmol/L |
| Umxube weProbe (10 μmol/L)b | × | 50 - 250 nmol/L |
| Idayi yereferensi yeRox | 0.5 | 1× |
| Isakhelo seDNA/cDNA | 1-10 | - |
| ddH2O | ukuya kuthi ga kwi-25 | - |
Amanqaku:Xuba ngokucokisekileyo phambi kokusetyenziswa ukunqanda amaqamza agqithisileyo ekungcangcazeleni ngamandla.
a.I-Primer concentration: I-Primer Mix iqulethe izibini ezininzi ze-primers, ngokuqhelekileyo i-primer nganye kwinqanaba lokugqibela le-0.2 μmol / L kwaye inokulungiswa phakathi kwe-0.1 kunye ne-0.5 μmol / L ngokufanelekileyo.
b.I-probe concentration: I-Probe Mix iqulethe iiprobes ezininzi ezineempawu ezahlukeneyo ze-fluorescence, kwaye ukuxinwa kweprobe nganye kunokulungiswa phakathi kwe-50 kunye ne-250 nmol / L ngokwemeko ethile.
1.Isalathiso sedayi ye-Rox:Isetyenziswa kwi-Real Time PCR isixhobo sokukhulisa ezifana ne-Applied Biosystems ukulungisa impazamo yomqondiso we-fluorescence owenziwe phakathi kwamaqula;le mveliso ayinayo isalathiso sedayi yeRox.I-Cas#10200 iyacetyiswa ukuba iyafuneka.
2.Template dilution: I-qPCR inovakalelo oluphezulu, kwaye kuyacetyiswa ukuba udibanise itemplate ukuze isetyenziswe.Ukuba ithemplate iyisisombululo sesitokhwe se-cDNA, umthamo wetemplate akufanele udlule i-1/10 yomthamo opheleleyo.
3.Inkqubo yokusabela: i-25μL, 30μL okanye i-50 μL iyacetyiswa ukuqinisekisa ukusebenza kunye nokuphindaphinda kwe-target gene yokukhulisa.
4.Ukulungiswa kwenkqubo: Nceda ulungiselele kwibhentshi ecocekileyo kakhulu, kwaye usebenzise iingcebiso kunye neetyhubhu zokusabela ngaphandle kwe-nuclease residu;kucetyiswa ukuba usebenzise iingcebiso ngeekhatriji zokucoca.Kuphephe ungcoliseko olunqamlezayo kunye nokungcoliseka kwe-aerosol.
2.Inkqubo yokusabela
| Inyathelo lomjikelo | Temp. | Ixesha | Imijikelo |
| I-denaturation yokuqala | 95 ℃ | 5 imiz | 1 |
| I-Denaturation | 95 ℃ | 15sec | 45 |
| Ukwandiswa/Ukwandiswa | 60 ℃ | 30sec |
Amanqaku:
1.I-Annealing / Extension: Iqondo lokushisa kunye nexesha linokulungelelaniswa ngokufanelekileyo ngokwexabiso le-primer eyilelwe i-Tm.
2.Ukufumana isignali ye-Fluorescence: Ixesha lokufumana isignali ye-fluorescence efunekayo kwizixhobo ezahlukeneyo zokubona i-qPCR yahlukile, nceda usethe ngokomda wexesha elincinci.Ixesha lezixhobo ezininzi eziqhelekileyo libekwe ngolu hlobo lulandelayo:
Imizuzwana ye-20: I-Biosystems esetyenzisiweyo 7700, 7900HT, 7500 Fast
31 imizuzwana: Biosystems esetyenziswayo 7300
Imizuzwana engama-32: I-Biosystems esetyenzisiweyo 7500
Amanqaku
Nceda unxibe iPPE eyimfuneko, idyasi yelebhu kunye neeglavu, ukuqinisekisa impilo nokhuseleko lwakho!
