RT-LAMP Fluorenscent Master mix
Iki gicuruzwa kirimo reaction ya reaction, RT-Enzymes ivanze (Bst ADN polymerase na Bris DNA irwanya ubushyuhe), Protectants ya lyophilized nafluorescentIbara.Buffer ya reaction irimo Mg2 +, dNTP nibindi bikoresho bikenewe kugirango amplification.Gukoresha, vanga gusa Buffer, reaction ya reaction, irangi rya fluorescent, primer hanyuma wongereho inyandikorugero, wongereho lyofilized protekant irashobora gukoresha neza.Yahujwe na lyophilizer na lyofilize, kandi primers na templates gusa byongeweho mugihe byakoreshejwe.Iyi reagent ifite ubushobozi bwo kongera imbaraga no kwiyumvisha ibintu.
Ibigize
| Ibigize | HCB5205A-01 | HCB5205A-02 | HCB5205A-03 |
| Umuyoboro uhuza Amplification Buffer | 1.2 mL | 4 mL × 3 | 12 mL × 10 |
| RT-Enzymes ivanze | 245 μL | 2.45 mL | 2.45 mL × 10 |
| Kurinda Lyophilized | 0,96 mL × 2 | 9.60 mL × 2 | 9.60 mL × 20 |
| Irangi | 192 μL | 0,96 mL × 2 | 0,96 mL × 20 |
Porogaramu
Kuri ADN cyangwa RNA isothermal amplification.
Ububiko
Gutwarwa na ice cyumye, kibitswe kuri -25 ~ -15 ℃.Irinde gukonjesha kenshi, ibicuruzwa bifite agaciro mumezi 12.
Porotokole
1.Kuramo buffer reaction kugirango ikoreshwe mubushyuhe bwicyumba.Vortex muri make cyangwa ihindure imiyoboro inshuro nyinshi kugirango ivange neza, hanyuma centrifuge kugirango ikusanyirize amazi munsi yigituba.
2.Gutegura sisitemu yo kwitwara.Iyi reagent irashobora gutegurwa muburyo bubiri bwa reaction, kuvanga ibintu bivanze no kuvanga sisitemu.
1) Tegura kuvanga ibintu bivanze
| Ibigize | Umubumbe |
| Umuyoboro uhuza Amplification Buffer | 12.5 μL |
| Irangi | 2 μL |
| RT-Enzymes ivanze | 2.55 μL |
| 10 mix Primer mixa | 5 μL |
| Inyandikorugero ADN / RNA b | × μL |
| Amazi adafite ingufu | Kugera kuri 50 μL |
2) Sisitemu yo kuvanga Lyophilisation
Tegura kuvanga lyofilize
| Ibigize | Umubumbe |
| Umuyoboro uhuza Amplification Buffer | 12.5 μL |
| Kurinda Lyophilized | 20 μL |
| Irangi | 2 μL |
| RT-Enzymes ivanze | 2.55 μL |
| Amazi adafite ingufu | Kugera kuri 50 μL |
Oph Lyophilisation: Ivanga ryateguwe ryakozwe muri sisitemu ya 50μL
Tegura kuvanga reaction
| Ibigize | Umubumbe |
| Kuvangavanga | Igice 1 |
| 10 mix Primer mixa | 5 μL |
| Inyandikorugero ADN / RNA b | × μL |
| Amazi adafite ingufu | Kugera kuri 50 μL |
Inyandiko:
1) a.10 × Primer mix: 16 μM FIP / BIP, 2 μM F3 / B3, 4 μM Umuzingi F / B;
2) b.DEPC (soluble water) irasabwa kuri nucleic aside templ.
3)Amplification Reaction: Shiraho gahunda ikurikira ku gikoresho cya fluorescent PCR (urugero: ABI7500, nibindi):
| Ubushyuhe | Igihe | Uruziga |
| 65 ℃ | 60 amasegonda * gukusanya FAM fluorescence | 30 |
Inyandiko
1.Umunyu urashobora kugaragara munsi yigituba cya buffer, vortex mugihe gito cyangwa guhinduranya imiyoboro inshuro nyinshi kugirango uvange neza mubushyuhe bwicyumba.
2.Ubushyuhe bwa reaction bushobora kuba bwiza hagati ya 62 ℃ na 68 ℃ ukurikije imiterere ya primers.
3.Ubushakashatsi bugomba kuba busanzwe, harimo gutegura sisitemu yo kubyitwaramo, lyophilisation, hamwe no gutunganya icyitegererezo hamwe no kongera icyitegererezo;
4.Kugira ngo wirinde kwanduza, birasabwa gutegura sisitemu yo kubyitwaramo mu ntebe isukuye cyane, mu zindi Ongeraho inyandikorugero kuri fume hood yicyumba kugirango wirinde kwivanga nabi.
