2×Sensi Direct Premix-UNG (Probe qPCR)
Nambala ya mphaka: HCB5151A
SensiDirect Premix-UNG (Probe qPCR) idapangidwa kuti izipanga PCR mwachindunji kuchokera ku zitsanzo popanda kutulutsa DNA kapena kukonzekera zitsanzo.Reagent iyi imakhala ndi DNA polymerase, uracil DNA glycosylase (UNG), RNase Inhibitor, MgCl.2, dNTPs (ndi dUTP m'malo mwa dTTP), ndi zokhazikika, za kuchuluka kwa PCR (qPCR).Reagent iyi imapangitsa kulolerana kwakukulu kwa inhibitor, motero imatha kugwiritsidwa ntchito mwachindunji pakuzindikira zitsanzo monga swab yapakhosi, malovu, anti-coagulated magazi athunthu, plasma, ndi seramu popanda kutulutsa DNA.The reagent imagwiritsa ntchito buffer ya qPCR yokhala ndi ma enzyme osakanikirana a DNA polymerase ndi UNG enzyme.Chifukwa chake, imatha kupeza kukulitsa bwino kwa majini omwe amatsata mu zitsanzo zomwe zili ndi zoletsa ndikuletsa kukulitsa kwabodza komwe kumachitika chifukwa cha kutsalira kwa PCR ndi kuipitsidwa kwa aerosol.Reagent iyi imagwirizana ndi zida zambiri za PCR za fulorosenti, monga Applied Biosystems, Eppendorf, Bio-Rad, Roche ndi zina zotero.
Zigawo
1. 50×SensiDirect Enzyme/UNG Mix
2. 2×SensiDirect 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.
Panjinga Protocol
| Khwerero | Kutentha | Nthawi | Kuzungulira |
| Kugaya chakudya | 50 ℃ | 2 min | 1 |
| Kutsegula kwa polymerase | 95 ℃ | 1-5 min | 1 |
| Denature | 95 ℃ | 10-20s | 40-50 |
| Zowonjezera / Zowonjezera | 56-64 ℃ | 20-60s |
Malangizo a Pipetting
| Reagent | Voliyumu pa anachita | Voliyumu pakuchita | Kukhazikika Kwambiri |
| 2×SensiDirect Premix Buffer (dUTP) | 12.5µL | 25µL | 1 × pa |
| 50×SensiDirect Enzyme/UNG Mix | 0.5µL | 1µl pa | 1 × pa |
| 25 × Primer-Probe Mix1, 2 | 1µl pa | 2µL | 1 × pa |
| Chitsanzo3, 4 | - | - | - |
| ddH2O | - | - | - |
| Voliyumu yonse | 25 μl pa | 50 μl pa | - |
1. Kuphatikizika komaliza kwa primer nthawi zambiri kumakhala 0.2μM.Kuti mupeze zotsatira zabwino, ndende yoyambira imatha kukonzedwa mkati mwa 0.2-1μM.
2. Nthawi zambiri, ndende ya kafukufukuyo imatha kukonzedwa mkati mwa 0.1-0.3μM.Kuchuluka kokwanira kwa kafukufukuyu kumakhudzana ndi nthawi yeniyeni yokulitsa chida cha PCR, mtundu wa kafukufuku, ndi mtundu wa zinthu zolembera za fulorosenti.Chonde onani buku la zida kapena zofunikira za kafukufuku wa fulorosenti iliyonse.
3. Mitundu yosiyanasiyana ya zitsanzo imakhala ndi mitundu yosiyanasiyana ndi zomwe zili mu inhibitor ndi chiwerengero cha jini chandamale.Voliyumu yachitsanzo iyenera kuganiziridwa ndi mkhalidwe weniweni.Pangani dilution ya chitsanzo powonjezera madzi opanda nuclease kapena TE Buffer, ngati kuli kofunikira.
4. Voliyumu yovomerezeka ya zitsanzo zosiyanasiyana:
| Chitsanzo | Voliyumu imodzi ya 50 μL anachita | Kuchuluka chiŵerengero |
| Anticoagulated magazi athunthu | 2.5 μL | 5% |
| Plasma | 15 μl pa | 30% |
| Seramu | 10 μl pa | 20% |
| Pakhosi pakhosi | 10 μl pa | 20% |
| Malovu | 10 μl pa | 20% |
Kuwongolera Kwabwino
1. Kuzindikira ntchito: kukhudzika, kukhazikika komanso kubwereza kwa qPCR.
2. Palibe ntchito ya nyukiliya yakunja: palibe endonuclease yachilendo komanso kuipitsidwa kwa exonuclease.
Zolemba Zamalonda
1. Izi zimagwiritsa ntchito mtundu wamtundu wa DNA polymerase wotentha, womwe ukhoza kutsegulidwa mu 1-5 minutes.Popeza momwe buffer yake yakonzedwa, ndiyoyenera kwambiri pawiri kapena angapo fulorosenti kachulukidwe PCR ntchito probe njira.
2. Ngati mtengo wa Rn wa kukulitsa kwa PCR ndi wotsika kwambiri kapena kukulitsa mwachiwonekere kumaletsedwa, kuchepetsa chiwerengero cha zitsanzo, kuwonjezereka kwa voliyumu kapena kuchepetsedwa kwachitsanzo kungapangitse zotsatira.
3. Kutoleredwa kwa magazi, malovu, mkodzo, swab yapakhosi, ndi zina zotero kuyenera kutsata zofunikira zachipatala, ndipo zitsanzo zatsopano zingagwiritsidwe ntchito poletsa kuwonongeka kwa nucleic acid.
4. Popeza ma amplicons osiyanasiyana ali ndi ntchito yosiyana yogwiritsira ntchito ku dUTP ndi kukhudzidwa kwa UNG, ma reagents ayenera kukonzedwa bwino ngati chidziwitso chodziwika chikuchepa pogwiritsa ntchito dongosolo la UNG.Chonde titumizireni thandizo laukadaulo ngati pakufunika.
5. Pofuna kupewa kukulitsa kwazinthu za PCR pakati pa zochita za gawo limodzi, malo oyesera odzipereka ndi pipette amafunikira pakukulitsa.Gwirani ntchito ndi magolovesi ndikusintha pafupipafupi ndipo musatsegule chubu chochita pambuyo pakukulitsa kwa PCR.
