(96-well) QuickEasy Cell Direct RT-qPCR Kit - SYBR Green I
Kufotokozera
The(96-chabwino) QuickEasyTM Cell Direct RT-qPCR Kit-SYBR Green Iamaperekadongosolo lapadera la lysis bufferto kumasula RNA mwachangukuchokera ku cell yotukukazitsanzo za machitidwe a RT-qPCR, kuchotsa nthawi yambiri komanso yotopetsaNjira yoyeretsera RNA, ndipo imangotenga mphindi 7 kuti mupeze template yofunikira ya RNA.The5 × Direct RT Mixndi2 × Mwachindunji qPCR Mix-SYBRzoperekedwa mu bokosi akhoza mwamsanga ndibwino kupeza zenizeni nthawi kachulukidweZotsatira za PCR.
5 × Direct RT Mix ndi 2 × Direct qPCR Mix-SYBR ali ndi kulekerera kwamphamvu kwa inhibitor, ndipo amatha kugwiritsa ntchito lysate yachitsanzo kuti ayesedwe ngati template kuti alembetse bwino ndikukulitsa.The reagent ili ndi Foregene yapadera Reverse Transcriptase yogwirizana kwambiri ndi RNA, komanso Hot D-Taq DNA Polymerase, dNTPs, MgCl.2 , chotchinga chochitira, PCR optimizer ndi stabilizer, ndipo chingagwiritsidwe ntchito pamodzi ndi lysis buffer kuti azindikire chitsanzocho mwamsanga, mosavuta komanso molondola, ndipo ali ndi makhalidwe okhudzidwa kwambiri, amphamvu kwambiri komanso okhazikika bwino.
Chidachi chimayang'ana pa micro-system lysis ya maselo otukuka bwino a 96, ndipo ali ndi mawonekedwe abwino komanso osasinthasintha;zigawo za zida zimapereka 96 lysis reactions, 96 reverse transcription reactions ndi 96 × 2 qPCR reactions, zomwe zimatha kukumana ndi mbale ya 96-well cell kuti igwiritse ntchito nthawi imodzi, kupeŵa kuipitsidwa komwe kumadza chifukwa chotsegula mobwerezabwereza, kuzizira ndi kusungunuka kwa reagents ndi kuwonongeka kwa ntchito ya reagent.
Zida zamagulu
| Kupanga kwa zida ( 50 μL lysis system/20 mlRT machitidwe /20μL qPCR reaction system) | Chithunzi cha DRT-03011 | Ndemanga | |
| 96t ndi | |||
| GawoI | BafaCL | 5ml pa | SeloLysis |
| ForegeneProtease Plus II | 100 μL | ||
| BafaST | 500 μL | ||
| Gawo II | DNAChofufutira | 100 μL | |
| 5 × Direct RT Mix | 400 μL | RT | |
| 2 × Direct qPCR Mix-SYBR | 1 mL × 2 pa | qPCR | |
| 50 × ROX Reference Dye | 400µL | ||
| RNase- KwaulereddH2 O | 1.7ml pa |
| |
| Manual | 1 chidutswa | 1 kutumikira | |
*: The lysis reagent DNA Eraser ikuphatikizidwa mu Gawo II la zida;Cell Lysis, RT, ndi zigawo za qPCR zitha kugulidwa padera.
Mbali & ubwino
■Zosavuta komanso zothandiza : ndiukadaulo wa Cell Direct RT, zitsanzo za RNA zitha kupezeka m'mphindi 7 zokha.
■ Zofuna zachitsanzo ndizochepa, mpaka ma cell 10 akhoza kuyesedwa.
■ Kutulutsa kwakukulu: kumatha kuzindikira mwachangu RNA m'maselo opangidwa mu 384, 96, 24, 12, 6-chitsime mbale.
■ DNA chofufutira akhoza kuchotsa mwamsanga matupi athu omasulidwa, kwambiri kuchepetsa zotsatira zoyeserera.
■ Makina okhathamiritsa a RT ndi qPCR amapangitsa kuti masitepe awiri a RT-PCR alembedwe bwino kwambiri komanso PCR yachindunji, komanso yosagwirizana ndi RT-qPCR reaction inhibitors.
Kit application
Kuchuluka kwa ntchito: maselo otukuka.
- RNA yotulutsidwa ndi zitsanzo za lysis: imagwira ntchito pa template ya RT-qPCR ya zida izi.
- Zidazi zitha kugwiritsidwa ntchito pazifukwa izi: kusanthula kwa jini, kutsimikizira kwa siRNA-mediated gene silence effect, kuyang'anira mankhwala, etc.
Kusungirako ndi moyo wa alumali
Gawo I la zida izi liyenera kusungidwa pa 4℃;Gawo II liyenera kusungidwa pa -20 ℃.
Foregene Protease Plus II iyenera kusungidwa pa 4℃, osazizira pa -20 ℃.
Reagent 2×Direct qPCR Mix-Taqman iyenera kusungidwa pa -20℃mumdima;ngati imagwiritsidwa ntchito pafupipafupi, imathanso kusungidwa pa 4℃ posungira kwakanthawi kochepa (gwiritsani ntchito mkati mwa masiku 10).
Real Time PCR primer design mfundo
Forward Primer ndi Reverse Primer
Kwa Real Time PCR, mapangidwe oyambira ndi ofunika kwambiri.Zoyambira zimayenderana ndi kutsimikizika komanso kuthekera kwa kukulitsa kwa PCR, ndipo zitha kupangidwa motengera mfundo izi:
- Kutalika koyambirira: 18-30bp.
- Zolemba za GC: 40-60%.
- Mtengo wa Tm: Mapulogalamu opangira ma primer, monga Primer 5, amatha kupereka mtengo wa Tm woyambira.Makhalidwe a Tm oyambira kumtunda ndi kumtunda ayenera kukhala pafupi momwe angathere.Njira yowerengera ya Tm ingagwiritsidwenso ntchito: Tm = 4 °C (G + C) + 2 °C (A + T).Pochita PCR, kutentha pansi pa mtengo woyambira wa Tm wa 5 ° C nthawi zambiri kumasankhidwa ngati kutentha kwapang'onopang'ono (kuwonjezeka kofananirako kwa kutentha kwapang'onopang'ono kumatha kukulitsa momwe PCR imachitira).
- Primers ndi zinthu za PCR:
- Design primer PCR amplification product kutalika makamaka 100-150bp.
- Zoyambira zojambula m'dera lachiwiri la template ziyenera kupewedwa momwe zingathere.
- Pewani kupanga zoyambira ziwiri kapena kupitilira apo pakati pa 3′ malekezero oyambira kumtunda ndi kumunsi kwa mtsinje.
- Primer 3′ terminal base sangakhalepo ndi 3 zowonjezera zotsatizana za G kapena C.
- Zoyambira zokha sizingakhale ndi zida zowonjezera, apo ayi mawonekedwe atsitsi adzapangidwa, zomwe zimakhudza kukulitsa kwa PCR.
- ATCG iyenera kugawidwa mofanana momwe mungathere poyambira, ndipo 3′ terminal base iyenera kupewedwa ngati T.
Zowonjezera1:Cndi DirectRT-qPCR Kit chigawot paketi yowonjezera
1.Cell Lysis Solution
| Cell Lysis Solution | |||
| Zida zamagulu (24-chitsime lysis dongosolo / bwino) | Chithunzi cha DRT-01011-A1 | Chithunzi cha DRT-01011-A2 | |
| 100 T | 500 T | ||
| GawoIne | Mtengo CL | 20 ml | 100 ml |
| Foregene Protease Plus II | 400 μl | 1 ml × 2 | |
| Mtengo wapatali wa magawo ST | 1 ml × 2 | 10 ml | |
| GawoII | DNA Eraser | 400 μl | 1 ml × 2 |
| RT Mix | |
| Zida zamagulu (20 μl reaction system) | Chithunzi cha DRT-01011-B1 |
| 200 T | |
| 5 × Direct RT Mix | 800 ml |
| RNase-Free ddH2O | 1.7ml × 2 |
| qPCR Mix | ||
| Zida zamagulu (20 μl reaction system) | DRT-01021-C1 | DRT-01021-C2 |
| 200 T | 1000 T | |
| 2 × Direct qPCR Mix-Taqman | 1 ml × 2 | 1.7ml × 6 |
| 20 × ROX Reference Dye | 40 ml | 200 μl |
| RNase-Free ddH2O | 1.7 ml | 10 ml |
Mabuku a Malangizo:
