Gawo limodzi RT-qPCR SYBR Green Premix
Nambala ya mphaka: HCB5140A
One Step RT-qPCR Syber Green Premix ndi ya fluorescence quantification kutengera utoto wa SYBR Green I.Pogwiritsa ntchito ma jini enieni oyambira, zolembera zosinthika ndi machitidwe a qPCR zimamalizidwa mu chubu chimodzi, kuchotsa kufunikira kotsegula kapu ndi kuyika mapaipi, kupititsa patsogolo luso la kuyesa ndikuchepetsa chiopsezo cha kuipitsidwa.Pazitsanzo za RNA, zidazo zimagwiritsa ntchito Reverse Transcriptase yosagwira kutentha kwa kaphatikizidwe kabwino ka cDNA ndi HotStart Taq DNA Polymerase pakukulitsa kuchuluka.Pansi pa makina okhathamiritsa, kukhudzika kwa zida kumatha kukhala kokwera mpaka 0.1 pg pazolinga zomwe zafotokozedwa kwambiri komanso mpaka 1 pg pazolinga zofotokozedwa bwino.Zidazi ndizoyenera kukulitsa ndi kuwerengetsa zitsanzo za DNA.Imathandizira kuzindikira ndikuwunika kwa ma nucleic acid kuchokera ku zitsanzo zosiyanasiyana za zomera ndi nyama, ma cell ndi tizilombo tating'onoting'ono.
Zigawo
No | Dzina | Voliyumu | Voliyumu |
1 | Advanced Buffer | 250 μl | 2 × 1.25 mL |
2 | Advanced Enzyme Mix | 20 μl pa | 200 μL |
3 | RNase Free H2O | 250 μl | 2 × 1.25 mL |
Zosungirako
Izi ziyenera kusungidwa pa -25 ~ -15 ℃ kutali ndi kuwala kwa 1 chaka.
Malangizo
1.Reaction dongosolo kasinthidwed
Zigawo | Kuchuluka (μL) | Kuchuluka (μL) | Kukhazikika komaliza |
Advanced Buffer | 12.5 | 25 | 1 × pa |
Advanced Enzyme Mix | 1 | 2 | - |
Woyambira Patsogolo (10 μmol/L)a | 0.5 | 1 | 0.2 μmol / L |
Choyambira Chosinthira (10 μmol/L)a | 0.5 | 1 | 0.2 μmol / L |
Chithunzi cha RNAb | X | X | - |
RNase Free H2Oc | ku 25 | ku 50 | - |
Ndemanga:
1) a.Tndende yomaliza yoyambira inali 0.2 μmol/L, yomwe imathanso kusinthidwa pakati pa 0.1 ndi 1μmol/L ngati kuli koyenera.
2) b.The reagent ndi tcheru kwambiri, ndi Total RNA mu osiyanasiyana 1pg-1μg, ndi kuyezetsa zitsanzo anthu anasonyeza mulingo woyenera kwambiri athandizira 1 pg-100 ng, kulamulira lonse Ct mtengo mu osiyanasiyana 15-30 monga koyenera.
3) c.Ndikofunikira kugwiritsa ntchito 20μL kapena 50μL kuti muwonetsetse kutsimikizika ndi kubwereza kwa jini yokulitsa.
4) d.Chonde konzekerani mu benchi yoyera kwambiri ndikugwiritsa ntchito maupangiri opanda zotsalira za nuclease ndi machubu ochitira;malangizo okhala ndi makatiriji fyuluta akulimbikitsidwa.Pewani kuipitsidwa ndi aerosol ndi kuipitsidwa.
2.Pulogalamu yamachitidwe
Sitepe yozungulira | Temp. | Nthawi | Zozungulira |
Sinthani zomasulira | 50 ℃a | 6 mins | 1 |
Denaturation koyamba | 95 ℃ | 5 mins | 1 |
Amplification reaction | 95 ℃ | 15 sec | 40 |
60 ℃b | 30 sec | ||
Gawo losungunuka | Zosasintha za Zida | 1 |
Ndemanga:
1) a.Kutentha kwa reverse transcript kumatha kusankhidwa pakati pa 50-55 ° C malinga ndi zosowa zoyesera.Kwa zitsanzo za DNA, njira yolembera m'mbuyo imatha kusiyidwa.
2) b.Muzochitika zapadera kutentha kwa annealing / kukulitsa kumatha kusinthidwa molingana ndi mtengo woyamba wa Tm, 60 ° C ndikulimbikitsidwa.
Zolemba
1. Izi ndizogwiritsidwa ntchito pofufuza kokha.
2. Chonde gwiritsani ntchito malaya a labu ndi magolovesi otayika, kuti mukhale otetezeka.