(96-maikaʻi) QuickEasy Cell Direct RT-qPCR Kit – SYBR Green I
Nā wehewehe
ʻO ka(96-well) QuickEasyTM Kelepona pololei RT-qPCR Kit–SYBR Green Ihoolakohe ʻōnaehana pale lysis kū hoʻokahito hoʻokuʻu koke i ka RNAmai ka moʻomeheu cellnā laʻana no nā pane RT-qPCR, e hoʻopau ana i ka manawa a me ka hanaʻO ke kaʻina hana hoʻomaʻemaʻe RNA, a lawe wale i 7 mau minuke e kiʻi i ka template RNA pono.ʻO ka5× Direct RT Mixa2× Direct qPCR Mix-SYBRhāʻawi ʻia i loko o ka pahu hiki koke aloaʻa maikaʻi ka quantitative manawa maoliNā hopena PCR.
ʻO 5 × Direct RT Mix a me 2 × Direct qPCR Mix-SYBR he ikaika ka hoʻomanawanui inhibitor, a hiki ke hoʻohana i ka lysate o ka hāpana e hoʻāʻo ʻia ma ke ʻano he kumu hoʻohālike no ka hoʻohuli ʻana i ka transcription a me ka hoʻonui kikoʻī.Loaʻa i ka reagent ka Foregene Reverse Transcriptase me ka pilina kiʻekiʻe no RNA, a me Hot D-Taq DNA Polymerase, dNTPs, MgCl2 , hoʻopaʻa hopena, PCR optimizer a stabilizer , a hiki ke hoʻohana pū ʻia me ka lysis buffer e ʻike i ka hāpana me ka wikiwiki, maʻalahi a pololei, a loaʻa iā ia nā ʻano o ka sensitivity kiʻekiʻe, ka kikoʻī ikaika a me ke kūpaʻa maikaʻi.
Hoʻoholo ʻia ka pahu i ka micro-system lysis o 96-well cultured cell, a loaʻa iā ia ka kūlike a me ke kūlike;Hāʻawi nā ʻāpana kit i 96 lysis reactions, 96 reverse transcription reactions a me 96 × 2 qPCR reactions, hiki ke hoʻokō i ka pā 96-well cell plate no ka hoʻohana ʻana i hoʻokahi manawa, e pale aku i ka pollution ma muli o ka wehe pinepine ʻana, ka hoʻoheheʻe ʻana a me ka hoʻoheheʻe ʻana o ka hana reagent.
Nā ʻāpana pahu
| haku mele ( 50 μL lysis system/20 μLRT ʻōnaehana pane /20μL qPCR ʻōnaehana pane) | DRT-03011 | ʻŌlelo | |
| 96T | |||
| MāheleI | HoʻopaʻaCL | 5 mL | KeleponaʻO Lysis |
| ForegeneProtease Plus II | 100 μL | ||
| HoʻopaʻaST | 500 μL | ||
| Māhele II | DNAHoloi | 100 μL | |
| 5× Direct RT Mix | 400 μL | RT | |
| 2× Direct qPCR Mix-SYBR | 1 mL × 2 | qPCR | |
| 50× ROX Mea Pepa Wai | 400µL | ||
| RNase- KuokoaddH2 O | 1.7mL |
| |
| Mmakahiki | 1 ʻāpana | 1 lawelawe | |
*: Hoʻokomo ʻia ka lysis reagent DNA Eraser ma ka Māhele II o ka pahu;Hiki ke kūʻai kaʻawale ʻia nā ʻāpana Cell Lysis, RT, a me qPCR.
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.
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 ka Direct qPCR Mix-Taqman 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.
Pākuʻi1:Cell DirectRT-qPCR ʻāpana pahut pūʻolo hoʻohui
1.Cell Lysis Solution
| ʻO ka hoʻonā cell Lysis | |||
| 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 |
| RT Mix | |
| 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 |
| Huina qPCR | ||
| Nā ʻāpana pahu (20 μl ʻōnaehana pane) | DRT-01021-C1 | DRT-01021-C2 |
| 200 T | 1000 T | |
| 2× Direct qPCR Mix-Taqman | 1 ml × 2 | 1.7 ml × 6 |
| 20× ROX Peʻa Waikane | 40 μl | 200 μl |
| RNase-Free ddH2O | 1.7 ml | 10 ml |
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