ISeli Ngqo RT qPCR Kit—Taqman Direct Cell Lysis Cell Ready Inyathelo elinye qRT-PCR Kits Probe
Iinkcazelo
Le mveliso isebenzisa inkqubo ye-lysis buffer ekhethekileyo yokukhulula ngokukhawuleza i-RNA kwiisampuli zeseli ezikhuliswe kwi-RT-qPCR ukuphendula, ukuphelisa ixesha elide kunye nenkqubo yokucoca i-RNA enzima, kunye nemizuzu ye-7 kuphela ukufumana i-template efunekayo ye-RNA, kunye ne-5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman ebonelelwa yikiti ye-quantitive ye-PC ngokukhawuleza
I-5 × Direct RT Mix kunye ne-2 × Direct qPCR Mix-Taqman inonyamezelo oluqinileyo lwe-inhibitor kwaye inokwenza ukuguqulwa okusebenzayo kunye nokukhulisa ngokuthe ngqo usebenzisa i-lysate yesampuli ukuba ilinganiswe njenge template.I-reagent iqulethe i-Foregene Reverse Transcriptase, i-Hot D-Taq DNA Polymerase, i-dNTPs, i-MgCl2, I-Reaction Buffer, i-PCR Optimizer kunye ne-Stabilizer, enokusetyenziswa kunye ne-lysis buffer ngokukhawuleza kwaye kulula ukubona iisampulu, kwaye ineempawu zobuntununtunu obuphezulu, ukuchaneka kunye nokuzinza.
Iinkcukacha
200×20μl Rxns, 1000×20μl Rxns
Amacandelo ekhithi
QuickEasyTMIseli ngqo RT-qPCR Kit-Taqman | ||||
Amacandelo ekhithi 20μl qPCR Reaction System | DRT-01021 | DRT-01022 | Phawula | |
200 T | 1000 T | |||
Inxalenye I | Isithinteli CL | 4 ml | 20 ml | Iseli Lysis |
Foregene Protease Plus II | 80 μl | 400 μl | ||
Isithinteli ST | 400 μl | 1 ml × 2 | ||
Inxalenye II | DNA Eraser | 80 μl | 400 μl | |
5×Ngqo RT uMxube * | 160 μl | 800 μl | RT | |
2× ngokuthe ngqo qPCR Mix-Taqman * | 1 ml × 2 | 1.7 ml × 6 | qPCR | |
I-20 × i-ROX ye-Reference Dye | 40 μl | 200 μl | ||
RNase-Free ddH2O | 1.7 ml | 10 ml | ||
Incwadi Yemiyalelo | Iqhekeza eli-1 | Iqhekeza eli-1 |
*:I-Cell Lysis, i-5 × Direct RT Mix, 2× Direct qPCR Mix-Taqman inokuthengwa ngokwahlukileyo
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.
Ugcino kunye nobomi beShelf
Inxalenye I yale khithi kufuneka igcinwe ngo-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-Taqman kufuneka igcinwe kwi -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.
Isihlomelo1:Cell ngqoI-RT-qPCR Ikhithi icandeloIpakethi yokongeza
1.Cell Lysis Solution
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 |
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 |
Umxube we-qPCR | ||
Amacandelo ekhithi (20 μl inkqubo yokusabela) | DRT-01021-C1 | DRT-01021-C2 |
200 T | 1000 T | |
2 × Direct qPCR Mix-Taqman | 1 ml × 2 | 1.7 ml × 6 |
I-20 × i-ROX ye-Reference Dye | 40 μl | 200 μl |
RNase-Free ddH2O | 1.7 ml | 10 ml |
Izikhokelo zoMyalelo: