Khwerero Chimodzi Mwachangu RT-qPCR Probe Premix-UNG
Nambala ya mphaka: HCR5143A
Gawo limodzi la RT-qPCR Probe Kit (FOR FAST) ndi chida chodziwira mwachangu cha RT-qPCR chomwe chili choyenera PCR ya single-plex kapena multiplex quantitative pogwiritsa ntchito RNA ngati template (monga kachilombo ka RNA).Izi zimagwiritsa ntchito m'badwo watsopano wa antibody-modified Taq DNA Polymerase ndi gawo limodzi lodzipatulira la Reverse Transcriptase, lokhala ndi chotchinga chokongoletsedwa chokulitsa mwachangu, chomwe chimakhala ndi liwiro lokulitsa, kuchita bwino kwambiri komanso kukhazikika.Imathandizira kukulitsa koyenera mu single-plex ndi multiplex ya zitsanzo zotsika komanso zapamwamba pakanthawi kochepa.
Zigawo
1.5×RT-qPCR buffer (U+)
2. Kusakaniza kwa enzyme (U+)
Ndemanga:
a.5×RT-qPCR buffer (U+) imaphatikizapo dNTP ndi Mg2+;
b.Kusakaniza kwa enzyme (U +) kumaphatikizapo reverse transcriptase, Hot Start Taq DNA polymerase, RNase inhibitor ndi UDG;
c.Gwiritsani ntchito malangizo a RNase-Free, machubu a EP, ndi zina.
Musanagwiritse ntchito, sakanizani bwino 5 × RT-qPCR buffer (U+).Ngati pali mvula ikatha kusungunuka, dikirani kuti bafa ibwerere kutentha kwa chipinda, sakanizani ndi kusungunuka, ndiyeno muzigwiritsa ntchito moyenera.
Zosungirako
Mankhwalawa amatumizidwa ndi ayezi wouma ndipo amatha kusungidwa ku -25 ~ -15 ℃ kwa chaka chimodzi.
Malangizo
1. Zimene anachita Dongosolo
| Zigawo | Kuchuluka (20 μL zomwe) |
| 2 × RT-qPCR buffer | 4 mul |
| Kusakaniza kwa enzyme (U+) | 0.8μL |
| Prime Forward | 0.1 ~ 1.0μM |
| Primer Reverse | 0.1 ~ 1.0μM |
| TaqMan Probe | 0.05 ~ 0.25μM |
| Template | X μL |
| Madzi Opanda RNase | mpaka 25 μl |
Zindikirani: Kuchuluka kwa mphamvu ndi 10-50μL.
2. Ndondomeko Yoyendetsa Panjinga (Swamba)
| Kuzungulira sitepe | Temp. | Nthawi | Zozungulira |
| Reverse Transcription | 55 ℃ | 10 min | 1 |
| Denaturation Yoyamba | 95 ℃ | 30 sec | 1 |
| Denaturation | 95 ℃ | 10 sec | 45 |
| Zowonjezera / Zowonjezera | 60 ℃ | 30 sec |
| Panjinga Protocol (Mwachangu) Kuzungulira sitepe |
Temp. |
Nthawi |
Zozungulira |
| Reverse Transcription | 55 ℃ | 5 min | 1 |
| Denaturation Yoyamba | 95 ℃ | 5 s | 1 |
| Denaturation | 95 ℃ | 3 s | 43 |
| Zowonjezera / Zowonjezera | 60 ℃ | 10 s |
Ndemanga:
a.Kutentha kwa reverse transcript kuli pakati pa 50 ℃ mpaka 60 ℃, kuwonjezera kutentha kumathandizira kukulitsa zida zovuta komanso ma tempuleti apamwamba a CG;
b.Kutentha koyenera kwa annealing kuyenera kusinthidwa kutengera mtengo wa Tm wa primer, ndikusankha nthawi yayifupi kwambiri yosonkhanitsira ma siginecha a fluorescence potengera chida cha Real Time PCR.
Zolemba
Chonde valani PPE yofunikira, malaya a labu ndi magolovesi, kuti mutsimikizire thanzi lanu ndi chitetezo!
