ʻO ke kelepona pololei RT qPCR Kit—SYBR GREEN I Direct Cell Lysis Cell Ready Hoʻokahi ʻanuʻu qRT-PCR Kits
Nā wehewehe
Hoʻohana kēia kit i kahi ʻōnaehana lysis buffer kūʻokoʻa e hiki ke hoʻokuʻu koke i ka RNA mai nā laʻana cell moʻomeheu no nā hopena RT-qPCR, a laila e hoʻopau i ke kaʻina hoʻomaʻemaʻe RNA hoʻopau manawa a paʻakikī.Hiki ke loaʻa ka template RNA i loko o 7 mau minuke.ʻO ka 5×Direct RT Mix a me 2×Hiki i nā reagents qPCR Mix-SYBR i hāʻawi ʻia e ka kit ke loaʻa koke i nā hopena PCR quantitative manawa maoli.
5×Direct RT Mix a me 2×Loaʻa i ka Direct qPCR Mix-SYBR ka ʻae ʻana i ka inhibitor ikaika, a hiki ke hoʻohana ʻia ka lysate o nā laʻana e like me ke kumu hoʻohālike no RT-qPCR pololei.Aia kēia pahu i ka RNA high-affinity Foregene reverse transcriptase, a me Hot D-Taq DNA polymerase, dNTPs, MgCl2, pale pale, PCR optimizer a stabilizer.
Nā kikoʻī
200×20μl Rxns, 1000×20μl Rxns
Nā ʻāpana pahu
| Mahele I | Hoʻopaʻa CL |
| Foregene Protease Plus II | |
| Hoʻopaʻa ST | |
| Mahele II | DNA Holoi |
| 5× Direct RT Mix | |
| 2× Direct qPCR Mix-SYBR | |
| 50× ROX Mea Pepa Wai | |
| RNase-Free ddH2O | |
| Nā kuhikuhi | |
Nā hiʻohiʻona & pono
■Maʻalahi a maikaʻi: me ka ʻenehana Cell Direct RT, hiki ke loaʻa nā laʻana RNA i loko o 7 mau minuke.
■ He liʻiliʻi ka noi hāpana, hiki ke hoʻāʻo ʻia ma kahi haʻahaʻa he 10.
■ Kiʻekiʻe throughput: hiki iā ia ke ʻike koke i ka RNA i loko o nā cell moʻomeheu i 384, 96, 24, 12, 6-well plates.
■ Hiki i ka DNA Eraser ke wehe koke i nā genome i hoʻokuʻu ʻia, e hoʻemi nui i ka hopena ma nā hopena hoʻokolohua ma hope.
■ ʻOi aku ka maikaʻi o ka ʻōnaehana RT-PCR i ʻoi aku ka maikaʻi a me ka PCR i ʻoi aku ka maikaʻi, a ʻoi aku ke kūpaʻa ʻana i nā mea hoʻopaneʻe hopena RT-qPCR.
Ke noi pahu
Ka laulā o ka hoʻohana ʻana: nā cell moʻomeheu.
- Hoʻokuʻu ʻia ʻo RNA e ka laʻana lysis: pili wale i ka template RT-qPCR o kēia pahu.
- Hiki ke hoʻohana ʻia ke kit no nā kumu aʻe: ka nānā ʻana i ka hōʻike ʻana o ke ʻano, ka hōʻoia o ka hopena silencing gene-mediated gene siRNA, ka nānā ʻana i ka lāʻau, etc.
Diagram
Ka mālama ʻana a me ka mālama ʻana
Pono e mālama ʻia ka ʻāpana I o kēia pahu ma 4 ℃;Pono e mālama ʻia ka ʻāpana II ma -20 ℃.
Pono e mālama ʻia ʻo Foregene Protease Plus II ma 4℃, mai maloʻo ma -20 ℃.
Mea hoʻoponopono 2×Pono e mālama ʻia ʻo Direct qPCR Mix-SYBR ma -20℃i ka pouli;inā hoʻohana pinepine ʻia, hiki ke mālama ʻia ma 4℃ no ka mālama manawa pōkole (hoʻohana i loko o 10 mau lā).
Nā loina hoʻolālā mua PCR manawa maoli
Kuhikuhi i mua a me ke kumu mua
No Real Time PCR, he mea nui ka hoʻolālā mua.Pili nā kumu mua i ka kikoʻī a me ka pono o ka hoʻonui ʻana i ka PCR, a hiki ke hoʻolālā ʻia me ka pili ʻana i nā kumu aʻe:
Ka lōʻihi mua: 18-30bp.
Maʻiʻo GC: 40-60%.
Waiwai Tm: Hiki i nā polokalamu hoʻolālā kumu, e like me Primer 5, ke hāʻawi i ka waiwai Tm o ke kumu.Pono e kokoke loa na koina Tm o na kumu mua a me lalo.Hiki ke hoʻohana ʻia ke kumu helu Tm: Tm = 4 °C (G + C) + 2 °C (A + T).Ke hana ʻia ka PCR, koho ʻia ka wela ma lalo o ka waiwai Tm kumu mua o 5 °C ma ke ʻano he wela annealing (hiki ke hoʻonui ʻia ka hoʻonui ʻana o ka mahana annealing i ka kikoʻī o ka hopena PCR).
Nā mea mua a me nā huahana PCR:
ʻOi aku ka maikaʻi o ka loaʻa ʻana o ka lōʻihi o ka huahana PCR amplification primer 100-150bp.
Pono e pale ʻia nā kumu mua o ka hoʻolālā ʻana ma ka ʻāpana kūkulu lua o ka template i ka mea hiki.
Hōʻalo i ka hoʻokumu ʻia ʻana o nā kumu hoʻohui ʻelua a ʻoi aʻe paha ma waena o nā welau 3′ o nā kumu mua a me lalo.
ʻAʻole hiki ke loaʻa ke kumu kumu kumu 3′ me 3 mau G a i ʻole C.
ʻAʻole hiki i nā primers ke loaʻa nā hale hoʻohui, inā ʻaʻole e hoʻokumu ʻia kahi ʻano lauoho lauoho, e pili ana i ka hoʻonui PCR.
Pono e puunaue like 'ia ka ATCG e like me ka hiki ma ke ka'ina mua, a e pale 'ia ke kumu 3' e like me T.
Pākuʻi 1: Pūʻulu hoʻohui ʻāpana ʻāpana o ka Pūnaewele RT-qPCR Kit
1.ʻO ka hoʻonā cell Lysis
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| Nā ʻāpana pahu (24-well lysis system / well) | DRT-01011-A1 | DRT-01011-A2 | |
| 100 T | 500 T | ||
| MāheleI | Hoʻopaʻa CL | 20 ml | 100 ml |
| Foregene Protease Plus II | 400 μl | 1 ml × 2 | |
| Hoʻopaʻa ST | 1 ml × 2 | 10 ml | |
| MāheleII | DNA Holoi | 400 μl | 1 ml × 2 |
2.RT Mix
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| Nā ʻāpana pahu (20 μl ʻōnaehana pane) | DRT-01011-B1 |
| 200 T | |
| 5× Direct RT Mix | 800 μl |
| RNase-Free ddH2O | 1.7 ml × 2 |
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| Nā ʻāpana pahu (20 μl ʻōnaehana pane) | DRT-01011-C1 | DRT-01011-C2 |
| 200 T | 1000 T | |
| 2× Direct qPCR Mix-SYBR | 1 ml × 2 | 1.7 ml × 6 |
| 50× ROX Mea Pepa Wai | 40 μl | 200 μl |
| RNase-Free ddH2O | 1.7 ml | 10 ml |
Nā Papa kuhikuhi:
