Akagari kayobora RT qPCR Kit-SYBR GREEN I Direct Cell Lysis Cell Yiteguye Intambwe imwe qRT-PCR Kits
Ibisobanuro
Iki gikoresho gikoresha sisitemu idasanzwe ya lysis buffer ishobora kurekura byihuse RNA mumikorere yimikorere yimikorere ya RT-qPCR, bityo bikuraho inzira yo kweza RNA itwara igihe kandi ikora cyane.Inyandikorugero ya RNA irashobora kuboneka muminota 7 gusa.5×Direct RT ivanze na 2×Direct qPCR Ivanga-SYBR reagent yatanzwe nigikoresho irashobora kwihuta kandi neza kubona ibisubizo nyabyo-byuzuye bya PCR.
5×Direct RT ivanze na 2×QPCR itaziguye ivanze-SYBR ifite kwihanganira imbaraga za inhibitor, kandi lysate yintangarugero irashobora gukoreshwa nkicyitegererezo cya RT-qPCR itaziguye.Iki gikoresho kirimo RNA idasanzwe-yuzuye ya Foregene reverse transcriptase, hamwe na polymerase ya D-Taq ishyushye, DNTPs, MgCl2, reaction ya reaction, optimizer ya PCR na stabilisateur.
Ibisobanuro
200×20μl Rxns, 1000×20μRxns
Ibigize ibikoresho
| Igice cya I. | Buffer CL |
| Foregene Protease Yongeyeho II | |
| Buffer ST | |
| Igice cya II | Gusiba ADN |
| 5 × Direct RT ivanze | |
| 2 × Direct qPCR ivanze-SYBR | |
| 50 × ROX Yerekana irangi | |
| RNase Yubusa ddH2O | |
| Amabwiriza | |
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.
Igishushanyo
Ububiko hamwe nubuzima bwa Shelf
Igice cya I cy'iki gikoresho 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-SYBR 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.
Umugereka 1 ： Akagari kayobora RT-qPCR Ibikoresho byuzuza ibikoresho
1.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 |
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 |
|
| ||
| Ibigize ibikoresho (20 μl sisitemu yo kwitwara) | DRT-01011-C1 | DRT-01011-C2 |
| 200 T. | 1000 T. | |
| 2 × Direct qPCR ivanze-SYBR | 1 ml × 2 | 1,7 ml × 6 |
| 50 × ROX Yerekana irangi | 40 μl | 200 μl |
| Ubusa ddH2O | 1,7 ml | 10 ml |
Imfashanyigisho:
Byihuta ByoroshyeTM Akagari Direct RT-qPCR Kit-SYBR Icyatsi I.
