Kuhle kakhulu Ukuqala, futhi I-Consumer Supreme iyisiqondiso sethu sokuletha izinsiza eziphezulu kubathengi bethu. Kulezi zinsuku, sizama konke okusemandleni ethu ukuba phakathi kwabathekelisi abaphezulu embonini yethu ukuze sihlangabezane nesidingo esikhulu sabathengi se-OEM Factory for High Fidelity Rt-Qpcr 2X One-Step Multiplex Master Mix, Sithembisa ukuzama okusemandleni ethu ukuze sikulethele imikhiqizo esezingeni eliphezulu nezomnotho nekhwalithi ephezulu.
Kuhle kakhulu Ukuqala, futhi I-Consumer Supreme iyisiqondiso sethu sokuletha izinsiza eziphezulu kubathengi bethu. Kulezi zinsuku, sizama konke okusemandleni ethu ukuba phakathi kwabathekelisi abaphezulu embonini yethu ukuze sihlangabezane nezidingo zabathengi ezengeziwe.I-China Taq DNA Polymerase ne-Qpcr, Ngesevisi ephakeme neyingqayizivele, sithuthukiswe kahle kanye namakhasimende ethu.Uchwepheshe nolwazi kuqinisekisa ukuthi sihlala sijabulela ukwethenjwa okuvela kumakhasimende ethu emisebenzini yethu yebhizinisi.“Ikhwalithi”, “ukwethembeka” kanye “nenkonzo” kuyisimiso sethu.Ukwethembeka kwethu nokuzibophezela kwethu kuhlala kunenhlonipho enkonzweni yakho.Xhumana nathi Namuhla Ukuze uthole imininingwane eyengeziwe, xhumana nathi manje.
Izimiso ze-Real Time PCR primer design

Dlulisela phambili i-Primer kanye ne-Primer Reverse

Nge-Real Time PCR, ukuklama kokuqala kubaluleke kakhulu.Ama-Primers ahlobene nokucaciswa nokusebenza kahle kokukhulisa i-PCR, futhi angaklanywa ngokubhekiselwa kule migomo elandelayo:

• Ubude be-Primer: 18-30bp.
• Okuqukethwe kwe-GC: 40-60%.
• Inani le-Tm: Isofthiwe yokuklama i-Primer, njenge-Primer 5, inganikeza inani le-Tm le-primer.Amanani e-Tm okuqalisa akhuphuka nomfula kufanele abe seduze ngangokunokwenzeka.Ifomula yokubala ye-Tm nayo ingasetshenziswa: Tm = 4 °C (G + C) + 2 °C (A + T).Lapho kwenziwa i-PCR, izinga lokushisa elingaphansi kwenani lokuqala le-Tm elingu-5 °C livame ukukhethwa njengezinga lokushisa lokukhipha isisu (ukwenyuka okuhambisanayo kwezinga lokushisa le-anneal kungakhuphula ukucaciswa kokusabela kwe-PCR).
• Ama-Primers kanye nemikhiqizo ye-PCR:

1. I-design primer PCR ubude bomkhiqizo wokukhulisa u-100-150bp.
2. Iziqalo zokuklama endaweni yesibili yesakhiwo sesifanekiso kufanele zigwenywe kakhulu ngangokunokwenzeka.
3. Gwema ukwakheka kwezisekelo ezi-2 noma ngaphezulu ezihambisanayo phakathi kwamaphethelo angu-3′ weziqalisi ezikhuphuka nomfula.
4. Isisekelo setheminali ye-Primer 3′ asikwazi ukuba khona nezinye ezingu-3 ezengeziwe ezilandelanayo ze-G noma C.
5. Ama-primer ngokwawo awakwazi ukuba nezakhiwo ezihambisanayo, ngaphandle kwalokho kuzokwakhiwa isakhiwo se-hairpin, esithinta ukukhuliswa kwe-PCR.
6. I-ATCG kufanele isatshalaliswe ngokulinganayo ngangokunokwenzeka ekulandeleni kokuqala, futhi isisekelo setheminali esingu-3′ kufanele sigwenywe njengo-T.

Isithasiselo1:Cethi DirectI-RT-qPCR Ingxenye yekhithit iphakethe le-supplement

1.Cell Lysis Solution

 Kuhle kakhulu Ukuqala, futhi I-Consumer Supreme iyisiqondiso sethu sokuletha izinsiza eziphezulu kubathengi bethu. Kulezi zinsuku, sizama konke okusemandleni ethu ukuba phakathi kwabathekelisi abaphezulu embonini yethu ukuze sihlangabezane nesidingo esikhulu sabathengi se-OEM Factory for High Fidelity Rt-Qpcr 2X One-Step Multiplex Master Mix, Sithembisa ukuzama okusemandleni ethu ukuze sikulethele imikhiqizo esezingeni eliphezulu nezomnotho nekhwalithi ephezulu.
OEM Factory forI-China Taq DNA Polymerase ne-Qpcr, Ngesevisi ephakeme neyingqayizivele, sithuthukiswe kahle kanye namakhasimende ethu.Uchwepheshe nolwazi kuqinisekisa ukuthi sihlala sijabulela ukwethenjwa okuvela kumakhasimende ethu emisebenzini yethu yebhizinisi.“Ikhwalithi”, “ukwethembeka” kanye “nenkonzo” kuyisimiso sethu.Ukwethembeka kwethu nokuzibophezela kwethu kuhlala kunenhlonipho enkonzweni yakho.Xhumana nathi Namuhla Ukuze uthole imininingwane eyengeziwe, xhumana nathi manje.

Izimiso ze-Real Time PCR primer design

Dlulisela phambili i-Primer kanye ne-Primer Reverse

Nge-Real Time PCR, ukuklama kokuqala kubaluleke kakhulu.Ama-Primers ahlobene nokucaciswa nokusebenza kahle kokukhulisa i-PCR, futhi angaklanywa ngokubhekiselwa kule migomo elandelayo:

• Ubude be-Primer: 18-30bp.
• Okuqukethwe kwe-GC: 40-60%.
• Inani le-Tm: Isofthiwe yokuklama i-Primer, njenge-Primer 5, inganikeza inani le-Tm le-primer.Amanani e-Tm okuqalisa akhuphuka nomfula kufanele abe seduze ngangokunokwenzeka.Ifomula yokubala ye-Tm nayo ingasetshenziswa: Tm = 4 °C (G + C) + 2 °C (A + T).Lapho kwenziwa i-PCR, izinga lokushisa elingaphansi kwenani lokuqala le-Tm elingu-5 °C livame ukukhethwa njengezinga lokushisa lokukhipha isisu (ukwenyuka okuhambisanayo kwezinga lokushisa le-anneal kungakhuphula ukucaciswa kokusabela kwe-PCR).
• Ama-Primers kanye nemikhiqizo ye-PCR:

1. I-design primer PCR ubude bomkhiqizo wokukhulisa u-100-150bp.
2. Iziqalo zokuklama endaweni yesibili yesakhiwo sesifanekiso kufanele zigwenywe kakhulu ngangokunokwenzeka.
3. Gwema ukwakheka kwezisekelo ezi-2 noma ngaphezulu ezihambisanayo phakathi kwamaphethelo angu-3′ weziqalisi ezikhuphuka nomfula.
4. Isisekelo setheminali ye-Primer 3′ asikwazi ukuba khona nezinye ezingu-3 ezengeziwe ezilandelanayo ze-G noma C.
5. Ama-primer ngokwawo awakwazi ukuba nezakhiwo ezihambisanayo, ngaphandle kwalokho kuzokwakhiwa isakhiwo se-hairpin, esithinta ukukhuliswa kwe-PCR.
6. I-ATCG kufanele isatshalaliswe ngokulinganayo ngangokunokwenzeka ekulandeleni kokuqala, futhi isisekelo setheminali esingu-3′ kufanele sigwenywe njengo-T.

Isithasiselo1:Cethi DirectI-RT-qPCR Ingxenye yekhithit iphakethe le-supplement

1.Cell Lysis Solution

Amamanuwali wemiyalo:

 I-Quick Easy Cell Direct RT-qPCR Kit-Taqman