(96-kakuhle) QuickEasy Cell Direct RT-qPCR Kit – Taqman
Iinkcazelo
I(96-kakuhle) QuickEasyTM ISeli Ngqo RT-qPCR Kit-Taqmaninikezainkqubo eyodwa ye-lysis bufferto khulula ngokukhawuleza i-RNAukusuka kwiseli culturediisampulu zeempendulo ze-RT-qPCR, ukuphelisa ixesha elitya kwaye linzimaInkqubo yokucoca i-RNA, kwaye ithatha imizuzu eyi-7 kuphela ukufumana ithemplate ye-RNA efunekayo.I5 × Ngqo RT Mixkwaye2 × Ngqo qPCR Mix-SYBRkubonelelwe kwibhokisi unako ngokukhawuleza kwayengokufanelekileyo ukufumana ubungakanani bexesha lokwenyaniIziphumo zePCR.
I-5 × Direct RT Mix kunye ne-2 × Direct qPCR Mix-Taqman inonyamezelo oluqinileyo lwe-inhibitor, kwaye inokusebenzisa i-lysate yesampuli ukuba ivavanywe njengethemplate ye-reverse transcription esebenzayo kunye nokukhulisa okuthile.I-reagent iqulethe i-Foregene unique Reverse Transcriptase enobudlelwane obuphezulu be-RNA, kunye ne-Hot D- Taq DNA Polymerase, i-dNTPs, i-MgCl2 , i-reaction buffer, i-PCR optimizer kunye ne-stabilizer, kwaye ingasetyenziselwa kunye ne-lysis buffer ukufumanisa isampuli ngokukhawuleza, ngokulula nangokuchanekileyo, kwaye ineempawu zobuntu obuphezulu, ukuchaneka okunamandla kunye nokuzinza okulungileyo.
Ikiti ijolise kwi-micro-system lysis ye-96-well cultured cells, kwaye inokufaniswa kakuhle kunye nokuhambelana;izixhobo zekhithi zibonelela nge-96 reactions ye-lysis, i-96 ye-reverse transcription reactions kunye ne-96 × 2 qPCR reactions, ezinokudibana ne-96-well cell plate ukuze zisetyenziswe ngexesha elinye, ukuphepha ukungcola okubangelwa ukuvulwa ngokuphindaphindiweyo, ukukhenkceza kunye nokunyibilika kwee-reagents kunye nokuthotywa kokusebenza kwe-reagent.
Amacandelo ekhithi
| Ukwakhiwa kweKit ( 50 μInkqubo ye-L lysis / 20 μLRT inkqubo yokusabela /20μInkqubo yokusabela ye-L qPCR) | DRT-03021 | Phawula | |
| 96T | |||
| InxalenyeI | IsithinteliCL | 5 ml | IseliULysis |
| I-ForegeneProtease Plus II | 100 μL | ||
| IsithinteliST | 500 μL | ||
| Inxalenye II | DNAIrabha | 100 μL | |
| 5 × Ngqo RT Mix | 400 μL | RT | |
| 2 × Ngqo qPCR Mix-Taqman | 1 mL × 2 | qPCR | |
| I-50 × i-ROX Reference Dye | 400µL | ||
| RNase- MahaladdH2 O | 1.7mL |
| |
| Munyaka | 1 iqhekeza | 1 ukukhonza | |
*: I-lysis reagent DNA Eraser ifakwe kwiSigaba II sekiti;I-Cell Lysis, i-RT, kunye namacandelo e-qPCR anokuthengwa ngokwahlukeneyo.
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:
