5 × Neoscript Fast RT-qPCR Premix kuphatikiza-UNG
Nambala ya mphaka: HCB5142A
Neoscript Fast RT Premix-UNG (Probe qRT-PCR) ndi chosakanikirana chokhazikika cha chubu chimodzi choyenera kulembedwa kwa gawo limodzi ndi kuchuluka kwa PCR (qRT-PCR).Imathandizira kusakanizikana kwa ma primers ndi ma probes ndikukhalabe okhazikika pambuyo posungira nthawi yayitali kutentha kochepa.Zitsanzo zomwe ziyenera kuyesedwa zitha kuwonjezeredwa mwachindunji mukamagwiritsa ntchito, popanda kutsegulira kwachubu / kuyika mapaipi.Izi zimapereka zigawo, mwachitsanzo, DNA polymerase yotentha, M-MLV, kutentha-labile uracil DNA glycosylase (TS-UNG), RNase Inhibitor, MgCl2, dNTPs (ndi dUTP m'malo mwa dTTP), ndi zokhazikika.Ndi ma genetic osinthidwa mwachangu amplification reverse transcriptase ndi DNA polymerase, ndizotheka kumaliza kukulitsa kwa PCR mkati mwa mphindi 20-40.Reagent iyi imagwiritsa ntchito chitetezo chapadera cha qPCR chokhala ndi ma enzyme osakanikirana a anti-inhibitory amplification enzyme ndi UNG enzyme.Chifukwa chake, imatha kupeza kukulitsa bwino kwa majini omwe mukufuna ndikupewa kukulitsa zabodza chifukwa cha kutsalira kwa PCR ndi kuyipitsidwa kwa aerosol.Reagent iyi imagwirizana ndi zida zambiri za PCR za fluorescence kuchokera kwa opanga monga Applied Biosystems, Eppendorf, Bio-Rad ndi Roche.
Chigawo
1.25×Neoscript Fast RTase/UNG Mix
2.5 × Neoscript Fast RT Premix Buffer (dUTP)
Zosungirako
Zigawo zonse ziyenera kusungidwa pa -20 ℃ posungira nthawi yayitali ndi 4 ℃ kwa miyezi itatu.Chonde sakanizani bwino mutatha kusungunuka ndi centrifuge musanagwiritse ntchito.Pewani kuzizira pafupipafupi.
Kukonzekera kwa qRT-PCR Reaction System
| Zigawo | 25μLDongosolo | 50μLDongosolo | Kukhazikika Kwambiri |
| 5 × Neoscript Fast RT Premix Buffer (dUTP) | 5 μl pa | 10 μl pa | 1 × pa |
| 25×Neoscript Fast RTase/UNG Mix | 1 μl pa | 2 mul | 1 × pa |
| 25 × Primer-Probe Mixa | 1 μl pa | 2 mul | 1 × pa |
| Chithunzi cha RNAb | - | - | - |
| ddH2O | Mpaka 25 μL | Mpaka 50μl | - |
1) a.Kuphatikizika komaliza kwa primer nthawi zambiri kumakhala 0.2μM.Kuti mupeze zotsatira zabwino, ndende yoyambira imatha kukonzedwa mkati mwa 0.2-1μM.Nthawi zambiri, ndende ya probe imatha kukonzedwa mkati mwa 0.1-0.3μM.
2) b.Pogwiritsa ntchito njira ya PCR yofulumira, kuonjezera kuchuluka kwa zoyambira ndi zofufuza kungapangitse zotsatira zabwino zokulitsa, ndipo chiŵerengero chawo chiyenera kukonzedwa moyenera.
3) Mitundu yosiyanasiyana ya zitsanzo imakhala ndi mitundu yosiyanasiyana komanso zomwe zili mu inhibitor komanso nambala ya jini yomwe mukufuna.Voliyumu yachitsanzo iyenera kuganiziridwa ndi mkhalidwe weniweni.Pangani dilution ya chitsanzo ndi madzi opanda nuclease kapena TE Buffer, ngati kuli kofunikira.
Reaction Czotsatira
| Ndondomeko ya PCR Yokhazikika | Fast PCR Njira | ||||||
| Ndondomeko | Temp. | Nthawi | Kuzungulira | Ndondomeko | Temp. | Nthawi | Kuzungulira |
| Reverse Transcription | 50 ℃ | 10-20mins | 1 | Reverse Transcription | 50 ℃ | 5 mins | 1 |
| Polymerase Kutsegula | 95 ℃ | 1-5 min | 1 | Polymerase Kutsegula | 95 ℃ | 30s | 1 |
| Denaturation | 95 ℃ | 10-20s | 40-50 | Denaturation | 95 ℃ | 1-3s | 40-50 |
| Annealing ndi Kuwonjezera | 56-64 ℃ | 20-60s | Annealing ndi Kuwonjezera | 56-64 ℃ | 3-20s | ||
Kuwongolera Kwabwino
1.Kuzindikira ntchito: kukhudzika, kutsimikizika ndi kubwereza kwa qPCR.
2.Palibe zochitika zamtundu wa nuclease: palibe endonuclease yachilendo komanso kuipitsa kwa exonuclease.
Zolemba
1.Kuchulukitsa kwa DNA polymerase mwachangu sikuchepera 1kb/10s.Zida zosiyanasiyana za PCR zimakhala ndi liwiro losiyanasiyana lotenthetsera ndi kuziziritsa, njira zowongolera kutentha ndi matenthedwe, motero kukhathamiritsa kwa primer / probe concentration ndi njira yothamanga kuphatikiza ndi chida chanu chachangu cha PCR ndikofunikira.
2.Chida ichi chimagwira ntchito mosiyanasiyana, ndipo ndichoyenera kuzindikiridwa ndi maselo amthupi.Njira ya PCR yamagawo atatu ndiyomwe ikulimbikitsidwa pazida zoyambira zokhala ndi kutentha pang'ono kapena kukulitsa zidutswa zazitali kupitilira 200 bp.
3.Popeza ma amplicons osiyanasiyana ali ndi magwiridwe antchito osiyanasiyana a dUTP komanso kukhudzika kosiyana kwa UNG, ma reagents ayenera kukonzedwa bwino ngati kuzindikira kucheperako mukamagwiritsa ntchito kachitidwe ka UNG.Chonde titumizireni thandizo laukadaulo ngati pakufunika.
4.Pofuna kupewa kukulitsa kwazinthu za PCR, malo oyesera odzipereka ndi pipette amafunikira pakukulitsa.Gwirani ntchito ndi magolovesi ndikusintha pafupipafupi ndipo musatsegule chubu cha PCR mutakulitsa.
