Cell Direct RT qPCR Kit—SYBR GREEN I Direct Cell Lysis Cell Ready One-step qRT-PCR Kits
Kufotokozera
Chidachi chimagwiritsa ntchito njira yapadera ya lysis buffer yomwe imatha kumasula mwachangu RNA kuchokera ku ma cell otukuka pamachitidwe a RT-qPCR, potero amachotsa njira yoyeretsera RNA yomwe imatenga nthawi komanso yovuta.The RNA template ikhoza kupezeka mu mphindi 7 zokha.The 5×Direct RT Mix ndi 2×Direct qPCR Mix-SYBR reagents zoperekedwa ndi zida zitha kupeza mwachangu komanso moyenera zotsatira za PCR zenizeni zenizeni.
5×Direct RT Mix ndi 2×Direct qPCR Mix-SYBR imakhala ndi kulekerera kwamphamvu kwa inhibitor, ndipo ma lysate a zitsanzo amatha kugwiritsidwa ntchito ngati template ya RT-qPCR mwachindunji.Chidachi chili ndi RNA yapamwamba kwambiri ya Foregene reverse transcriptase, ndi Hot D-Taq DNA polymerase, dNTPs, MgCl.2, zotchingira zochita, PCR optimizer ndi stabilizer.
Zofotokozera
200×20μl Rxn, 1000×20μl rxns
Zida zamagulu
| Gawo I | Mtengo CL |
| Foregene Protease Plus II | |
| Mtengo wapatali wa magawo ST | |
| Gawo II | DNA Eraser |
| 5 × Direct RT Mix | |
| 2 × Mwachindunji qPCR Mix-SYBR | |
| 50 × ROX Reference Dye | |
| RNase-Free ddH2O | |
| Malangizo | |
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.
Chithunzi
Kusungirako ndi moyo wa alumali
Gawo I la zida izi ziyenera 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-SYBR 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.
Zowonjezera 1: Cell Direct RT-qPCR Kit chigawo chowonjezera paketi
1.Cell Lysis Solution
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| |||
| 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 |
2.RT Mix
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| |
| 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 |
|
| ||
| Zida zamagulu (20 μl reaction system) | DRT-01011-C1 | DRT-01011-C2 |
| 200 T | 1000 T | |
| 2 × Mwachindunji qPCR Mix-SYBR | 1 ml × 2 | 1.7ml × 6 |
| 50 × ROX Reference Dye | 40 ml | 200 μl |
| RNase-Free ddH2O | 1.7 ml | 10 ml |
Mabuku a Malangizo:
