Inhlangano iqhubeka nomqondo wenqubo othi “ukuphathwa kwesayensi, ukubaluleka okuphezulu nokusebenza kahle, umthengi ophakeme we-China Manufacturer we-2× Concentrated Premix ye-Unpurified Sample Real-Time Quantitative PCR Direct Multiplex Probe Qpcr Mix Plus U, Ibhizinisi lethu lizinikezele ekunikezeni amakhasimende imikhiqizo ebalulekile nevikelekile yekhwalithi ephezulu ngentengo ngayinye enolaka futhi eqinile.
Inhlangano iqhubeka nomqondo wenqubo othi “ukuphathwa kwesayensi, ikhwalithi ephezulu kanye nokusebenza kahle okukhulu, umthengi ophakemeI-China Taq DNA Polymerase ne-Qpcr, Inkampani yethu inamandla amaningi futhi inohlelo lwenethiwekhi yokuthengisa oluzinzile noluphelele.Sifisa sengathi singasungula ubudlelwano obuhle bebhizinisi nawo wonke amakhasimende asekhaya nakwamanye amazwe ngesisekelo sezinzuzo ezifanayo.
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

 Inhlangano iqhubeka nomqondo wenqubo othi “ukuphathwa kwesayensi, ukubaluleka okuphezulu nokusebenza kahle, umthengi ophakeme we-China Manufacturer we-2× Concentrated Premix ye-Unpurified Sample Real-Time Quantitative PCR Direct Multiplex Probe Qpcr Mix Plus U, Ibhizinisi lethu lizinikezele ekunikezeni amakhasimende imikhiqizo ebalulekile nevikelekile yekhwalithi ephezulu ngentengo ngayinye enolaka futhi eqinile.
China Umkhiqizi weI-China Taq DNA Polymerase ne-Qpcr, Inkampani yethu inamandla amaningi futhi inohlelo lwenethiwekhi yokuthengisa oluzinzile noluphelele.Sifisa sengathi singasungula ubudlelwano obuhle bebhizinisi nawo wonke amakhasimende asekhaya nakwamanye amazwe ngesisekelo sezinzuzo ezifanayo.

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