Akagari kayobora RT qPCR Kit-Taqman Direct Cell Lysis Akagari Yiteguye Intambwe imwe qRT-PCR Kits Probe
Ibisobanuro
Iki gicuruzwa gikoresha sisitemu idasanzwe ya lysis kugirango irekure vuba RNA ingero zumuco zumuco kubitekerezo bya RT-qPCR, bivanaho uburyo bwo kweza RNA butwara igihe kandi bukora cyane, niminota 7 gusa kugirango ubone icyitegererezo cya RNA gisabwa, hamwe na 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman yatanzwe nigitabo irashobora kubona vuba kandi neza ibisubizo byuzuye bya PCR.
5 × Direct RT ivanze na 2 × Direct qPCR Ivanga-Taqman ifite kwihanganira imbaraga za inhibitor kandi irashobora gukora neza kandi igahinduka ikoresheje lysate yicyitegererezo igomba gupimwa nkicyitegererezo.Reagent irimo Foregene Reverse Transcriptase, Hot D-Taq ADN Polymerase, DNTPs, MgCl2, Buffer Reaction, PCR Optimizer na Stabilizer, irashobora gukoreshwa na lysis buffer kugirango byihuse kandi byoroshye kumenya ingero, kandi ifite ibiranga sensibilité yo hejuru, umwihariko kandi uhamye.
Ibisobanuro
200×20μl Rxns, 1000×20μRxns
Ibigize ibikoresho
| ByihuseTMAkagari kayobora RT-qPCR Kit-Taqman | ||||
| Ibigize ibikoresho 20μl qPCR Sisitemu yo Kwitwara | DRT-01021 | DRT-01022 | Icyitonderwa | |
| 200 T. | 1000 T. | |||
|
Igice I | Buffer CL | 4 ml | 20 ml | Lysis |
| Foregene Protease Yongeyeho II | 80 μl | 400 μl | ||
| Buffer ST | 400 μl | 1 ml × 2 | ||
|
Igice II | Gusiba ADN | 80 μl | 400 μl | |
| 5 × Direct RT ivanze * | 160 μl | 800 μl | RT | |
| 2 × Direct qPCR Ivanga-Taqman * | 1 ml × 2 | 1,7 ml × 6 | QPCR | |
| 20 × Irangi ryerekana irangi | 40 μl | 200 μl | ||
| Ubusa ddH2O | 1,7 ml | 10 ml | ||
| Igitabo gikubiyemo amabwiriza | Igice 1 | Igice 1 | ||
*:Cell Lysis, 5 × Direct RT ivanze, 2 × Direct qPCR ivanze-Taqman irashobora kugurwa ukwayo
Ibiranga ibyiza
■Byoroshye kandi byiza: hamwe na tekinoroji ya Cell Direct RT, ingero za RNA zirashobora kuboneka muminota 7 gusa.
Icyitegererezo gikenewe ni gito, nkuko selile 10 zishobora kugeragezwa.
Thr Kwinjiza cyane: irashobora kumenya vuba RNA mu ngirabuzimafatizo zifite 384, 96, 24, 12, 6-amasahani meza.
Er ADN Eraser irashobora gukuraho vuba genome zasohotse, bikagabanya cyane ingaruka kubisubizo byakurikiyeho.
System Optimized RT na qPCR sisitemu ituma intambwe ebyiri RT-PCR ihinduranya transcript neza kandi PCR ikarushaho kuba nziza, kandi ikarwanya RT-qPCR inhibitor.
Porogaramu
Igipimo cyo gushyira mu bikorwa: ingirabuzimafatizo.
- RNA yarekuwe na sample lysis: ikoreshwa gusa kuri RT-qPCR inyandikorugero yiki gitabo.
- Igikoresho kirashobora gukoreshwa mubikorwa bikurikira: gusesengura imvugo ya gene, kugenzura ingaruka zo gucecekesha gene ya siRNA, gusuzuma ibiyobyabwenge, nibindi.
Ububiko hamwe nubuzima bwa Shelf
Igice cya I cyiki gitabo kigomba kubikwa kuri 4℃;Igice cya II kigomba kubikwa kuri -20 ℃.
Foregene Protease Plus II igomba kubikwa kuri 4, Ntugahagarike kuri -20 ℃.
Reagent 2×QPCR itaziguye ivanze-Taqman igomba kubikwa kuri -20℃mu mwijima;iyo ikoreshejwe kenshi, irashobora kandi kubikwa kuri 4Kubika igihe gito (koresha muminsi 10).
Igihe nyacyo PCR primer igishushanyo mbonera
Imbere Primer na Reverse Primer
Kubihe Byukuri PCR, igishushanyo mbonera ni ngombwa cyane.Ibanze bifitanye isano nuburyo bwihariye bwo kongera PCR, kandi birashobora gushushanywa hifashishijwe amahame akurikira:
- Uburebure bwa primer: 18-30bp.
- Ibirimo GC: 40-60%.
- Agaciro ka Tm: Porogaramu ishushanya, nka Primer 5, irashobora gutanga Tm agaciro ka primer.Indangagaciro za Tm zo hejuru no kumanuka primers zigomba kuba hafi bishoboka.Inzira yo kubara ya Tm irashobora kandi gukoreshwa: Tm = 4 ° C (G + C) + 2 ° C (A + T).Iyo ukora PCR, ubushyuhe buri munsi ya primer Tm agaciro ka 5 ° C muri rusange byatoranijwe nkubushyuhe bwa annealing (kwiyongera gukwiranye nubushyuhe bwa annealing birashobora kongera umwihariko wa reaction ya PCR).
- Ibicuruzwa nibicuruzwa bya PCR:
- Gushushanya primer PCR yongerera ibicuruzwa uburebure nibyiza 100-150bp.
- Igishushanyo mbonera mugice cya kabiri cyubatswe cyicyitegererezo kigomba kwirindwa bishoboka.
- Irinde gushiraho ibice 2 cyangwa byinshi byuzuzanya hagati ya 3 ′ impera yimbere na epfo na ruguru.
- Primer 3 ′ itumanaho ntirishobora kuboneka hamwe na 3 yongeyeho G cyangwa C.
- Primers ubwayo ntishobora kugira ibyuzuzanya, bitabaye ibyo imiterere yimisatsi izashyirwaho, bigira ingaruka kuri PCR.
- ATCG igomba gukwirakwizwa uko bishoboka kwose murwego rwa primer, kandi 3 ′ base base igomba kwirindwa nka T.
Umugereka1 ： C.ellRT-qPCR Kit component
1.Cell Lysis Igisubizo
| Akagari ka Lysis | |||
| Ibigize ibikoresho (24-neza lysis sisitemu / neza) | DRT-01011-A1 | DRT-01011-A2 | |
| 100 T. | 500 T. | ||
| IgiceI. | Buffer CL | 20 ml | 100 ml |
| Foregene Protease Yongeyeho II | 400 μl | 1 ml × 2 | |
| Buffer ST | 1 ml × 2 | 10 ml | |
| IgiceII | Gusiba ADN | 400 μl | 1 ml × 2 |
| RT Kuvanga | |
| Ibigize ibikoresho (20 μl sisitemu yo kwitwara) | DRT-01011-B1 |
| 200 T. | |
| 5 × Direct RT ivanze | 800 μl |
| Ubusa ddH2O | 1,7 ml × 2 |
| QPCR Kuvanga | ||
| Ibigize ibikoresho (20 μl sisitemu yo kwitwara) | DRT-01021-C1 | DRT-01021-C2 |
| 200 T. | 1000 T. | |
| 2 × Direct qPCR Ivanga-Taqman | 1 ml × 2 | 1,7 ml × 6 |
| 20 × Irangi ryerekana irangi | 40 μl | 200 μl |
| Ubusa ddH2O | 1,7 ml | 10 ml |
Imfashanyigisho:
