ISeli Ngqo RT qPCR Kit—SYBR GREEN I Direct Cell Lysis Cell Ready Inyathelo elinye qRT-PCR Kits
Iinkcazelo
Le khithi isebenzisa inkqubo ye-lysis buffer ekhethekileyo enokuthi ikhulule ngokukhawuleza i-RNA kwiisampulu zeseli ezikhuliswe kwiimpendulo ze-RT-qPCR, ngaloo ndlela iphelisa inkqubo yokucoca i-RNA ethatha ixesha kunye nenzima.Ithemplethi ye-RNA inokufumaneka kwimizuzu nje esi-7.I-5×Ngqo RT Mix kunye 2×Iirejenti ezithe ngqo ze-qPCR Mix-SYBR ezibonelelwe yikhithi zinokukhawuleza nangempumelelo ukufumana iziphumo zePCR zexesha lokwenyani.
5×Ngqo RT Mix kunye 2×Direct qPCR Mix-SYBR babe inhibitor unyamezelo eyomeleleyo, kunye lysate iisampuli zingasetyenziswa njenge template RT-qPCR ngqo.Le khithi iqulethe i-RNA ephezulu ehambelana ne-Foregene reverse transcriptase, kunye ne-Hot D-Taq DNA polymerase, i-dNTPs, i-MgCl2, i-reaction buffer, i-PCR optimizer kunye ne-stabilizer.
Iinkcukacha
200×20μl Rxns, 1000×20μl Rxns
Amacandelo ekhithi
| Icandelo I | Isithinteli CL |
| Foregene Protease Plus II | |
| Isithinteli ST | |
| Icandelo II | DNA Eraser |
| 5 × Ngqo RT Mix | |
| 2 × Ngqo qPCR Mix-SYBR | |
| I-50 × i-ROX Reference Dye | |
| RNase-Free ddH2O | |
| Imiyalelo | |
Iimpawu&izinto eziluncedo
■Ilula kwaye iyasebenza : ngeTekhnoloji ye-Cell Direct RT, iisampuli ze-RNA zinokufumaneka kwimizuzu eyi-7 nje.
∎ Imfuno yesampulu incinci, ingaphantsi njengeeseli ezili-10 zinokuvavanywa.
■ Ukugqithisa okuphezulu: inokubona ngokukhawuleza i-RNA kwiiseli ezikhuliswe kwii-384, 96, 24, 12, 6-well plates.
■ I-DNA Eraser inokususa ngokukhawuleza iigenomes ezikhutshiweyo, inciphise kakhulu impembelelo kwiziphumo zovavanyo ezilandelayo.
■ Inkqubo elungiselelwe i-RT kunye ne-qPCR yenza amanyathelo amabini e-RT-PCR akhutshelwe umva asebenze ngakumbi kwaye i-PCR icace ngakumbi, kwaye imelane ngakumbi ne-RT-qPCR reaction inhibitors.
Isicelo seKit
Ububanzi besicelo: iiseli ezikhuliswe.
- I-RNA ekhutshwe ngesampuli ye-lysis: isebenza kuphela kwi-template ye-RT-qPCR yale khithi.
- Ikiti ingasetyenziselwa ezi njongo zilandelayo: uhlalutyo lwe-gene expression, ukuqinisekiswa kwe-siRNA-mediated gene silence effect, ukuhlolwa kweziyobisi, njl.
Umzobo
Ugcino kunye nobomi beShelf
Inxalenye I yale khithi kufuneka igcinwe kwi-4℃;Icandelo II kufuneka ligcinwe kwi -20 ℃.
I-Foregene Protease Plus II kufuneka igcinwe ngo-4℃, musa ukuba ngumkhenkce ku -20℃.
I-Reagent 2×Direct qPCR Mix-SYBR kufuneka igcinwe -20℃ebumnyameni;ukuba isetyenziswa rhoqo, inokugcinwa ngo-4℃ kugcino lwexesha elifutshane (sebenzisa ngaphakathi kweentsuku ezili-10).
Ixesha lokwenyani imigaqo yoyilo lwe-PCR primer
I-Primer yokuPhambili kunye ne-Primer yokuBuyisa
Ngexesha lokwenyani iPCR, uyilo lweprimer lubaluleke kakhulu.Iiprimers zinxulumene nobucacileyo kunye nokusebenza kakuhle kokwandiswa kwePCR, kwaye zinokuyilwa ngokubhekiselele kule migaqo ilandelayo:
Ubude bePrimer: 18-30bp.
Umxholo weGC: 40-60%.
Ixabiso le-Tm: I-software yoyilo lwe-Primer, njenge-Primer 5, inokunika ixabiso le-Tm le-primer.Amaxabiso e-Tm ee-primers ezinyukayo nezisezantsi kufuneka zibe kufutshane kangangoko.Ifomula yokubala ye-Tm nayo ingasetyenziswa: Tm = 4 °C (G + C) + 2 °C (A + T).Xa kusenziwa i-PCR, iqondo lobushushu elingaphantsi kwexabiso le-primer Tm le-5 °C likhethwa ngokubanzi njengeqondo lobushushu lokutshisa (ukunyuka okuhambelanayo kwiqondo lobushushu lobushushu kunokunyusa ukuchana kwe-PCR reaction).
Iiprimers kunye neemveliso zePCR:
I-design primer PCR yokukhulisa ubude bemveliso ikhethwa yi-100-150bp.
Ii-primers zoyilo kwindawo yesibini yesakhiwo setemplate kufuneka igwenywe kangangoko kunokwenzeka.
Gwema ukubunjwa kweziseko ezi-2 okanye ngaphezulu ezihambelanayo phakathi kwee-3′ zeziphelo ze-primers ezinyukayo kunye nezantsi.
Isiseko se-Primer 3′ asikwazi ukubakho nge-3 eyongezelelweyo elandelelanayo i-G okanye i-C.
Iiprimers ngokwazo azikwazi ukuba nezakhiwo ezincedisayo, ngaphandle koko kuya kwenziwa isakhiwo se-hairpin, esichaphazela ukukhulisa i-PCR.
I-ATCG kufuneka isasazwe ngokulinganayo ngokulandelelana kwe-primer, kwaye isiseko se-terminal ye-3 kufuneka sigwenywe njenge-T.
ISihlomelo 1: Ipakethi yecandelo leCell Direct RT-qPCR Kit
1.Iseli Lysis Solution
|
| |||
| Amacandelo ekhithi (Inkqubo ye-lysis ye-24 / kakuhle) | DRT-01011-A1 | DRT-01011-A2 | |
| 100 T | 500 T | ||
| InxalenyeI | Isithinteli CL | 20 ml | 100 ml |
| Foregene Protease Plus II | 400 μl | 1 ml × 2 | |
| Isithinteli ST | 1 ml × 2 | 10 ml | |
| InxalenyeII | DNA Eraser | 400 μl | 1 ml × 2 |
2.RT Umxube
|
| |
| Amacandelo ekhithi (20 μl inkqubo yokusabela) | DRT-01011-B1 |
| 200 T | |
| 5 × Ngqo RT Mix | 800 μl |
| RNase-Free ddH2O | 1.7 ml × 2 |
|
| ||
| Amacandelo ekhithi (20 μl inkqubo yokusabela) | DRT-01011-C1 | DRT-01011-C2 |
| 200 T | 1000 T | |
| 2 × Ngqo qPCR Mix-SYBR | 1 ml × 2 | 1.7 ml × 6 |
| I-50 × i-ROX Reference Dye | 40 μl | 200 μl |
| RNase-Free ddH2O | 1.7 ml | 10 ml |
Izikhokelo zoMyalelo:
