I-2×Sensi Direct Premix-UNG (Probe qPCR)
Inombolo yekati: HCB5151A
I-SensiDirect Premix-UNG (Probe qPCR) yenzelwe ukwenza i-PCR ngokuthe ngqo kwiisampuli ngaphandle kokutsalwa kwe-DNA okanye ukulungiswa kwesampulu.Esi sixhobo siqukethe i-DNA polymerase eshushu, uracil DNA glycosylase (UNG), RNase Inhibitor, MgCl.2, ii-dNTPs (kunye ne-dUTP endaweni ye-dTTP), kunye nezinzisi, ze-PCR yobungakanani (qPCR).Esi sixhobo siqinisekisa ukunyamezela okuphezulu kwe-inhibitor, kwaye ke sinokusetyenziswa ngokuthe ngqo ekubhaqweni kweesampulu ezinje ngeswab yomqala, amathe, igazi elichasene ne-coagulated lonke, iplasma, kunye neserum ngaphandle kokutsalwa kwe-DNA.I-reagent isebenzisa i-buffer yobunini ye-qPCR kunye ne-enzymes edibeneyo ye-anti-inhibitory DNA polymerase kunye ne-UNG enzyme.Ke ngoko, inokufumana ulwandiso olulungileyo lwejene ekujoliswe kuyo kwiisampulu eziqulethe inhibitors kwaye inqanda ukwandiswa kobuxoki okubangelwa yintsalela yePCR kunye nongcoliseko lwe-aerosol.Le reagent iyahambelana nezixhobo ezininzi ze-PCR ze-fluorescent, ezifana ne-Applied Biosystems, i-Eppendorf, i-Bio-Rad, i-Roche njalo njalo.
Amacandelo
1. 50×SensiDirect Enzyme/UNG Mix
2. 2×SensiDirect Premix Buffer (dUTP)
Iimeko zokugcina
Onke amacandelo kufuneka agcinwe ku -20℃ ukulungiselela ukugcinwa kwexesha elide kunye ne-4℃ ukuya kwiinyanga ezi-3.Nceda udibanise kakuhle emva kokunyibilika kunye ne-centrifuge ngaphambi kokusebenzisa.Kuphephe ukunyibilika rhoqo komkhenkce.
Umgaqo weBhayisekile
| Inyathelo | Ubushushu | Ixesha | Umjikelo |
| Ukwetyisa | 50℃ | 2 min | 1 |
| Ukusebenza kwePolymerase | 95℃ | 1-5min | 1 |
| Denature | 95℃ | 10-20s | 40-50 |
| Ukwandiswa/Ukwandiswa | 56-64℃ | 20-60s |
Imiyalelo yokufaka imibhobho
| I-Reagent | Umthamo ngokwe ukusabela | Umthamo ngokwempendulo | Ukugxininiswa kokugqibela |
| 2×SensiDirect Premix Buffer (dUTP) | 12.5µL | 25µL | 1× |
| 50×SensiDirect Enzyme/UNG Mix | 0.5µL | 1µL | 1× |
| 25×Primer-Probe Mix1, 2 | 1µL | 2µL | 1× |
| Isampulu3, 4 | - | - | - |
| ddH2O | - | - | - |
| Umthamo opheleleyo | 25 μL | 50 μL | - |
1. Ukugxininiswa kokugqibela kwe-primer ngokuqhelekileyo ngu-0.2μM.Ukufumana iziphumo ezingcono, ugxininiso lwe-primer lunokuphuculwa phakathi koluhlu lwe-0.2-1μM.
2. Ngokuqhelekileyo, i-concentration ye-probe inokuphuculwa ngaphakathi koluhlu lwe-0.1-0.3μM.Olona xinaniso lweprobe lunxulumene nexesha lokwenyani lesixhobo sokukhulisa iPCR, udidi lweprobe, kunye nodidi lwento yokuleyibhela ngefluorescent.Nceda ubhekisele kwincwadana yemigaqo yesixhobo okanye kwiimfuno ezikhethekileyo zeprobe nganye yefluorescent.
3. Iindidi ezahlukeneyo zeesampulu ziqulethe iintlobo ezahlukeneyo kunye nomxholo we-inhibitor kunye nekopi yekopi ye-gene ekujoliswe kuyo.Umthamo wesampuli kufuneka uqwalaselwe yimeko yangempela.Yenza i-dilution yesampuli ngokudibanisa amanzi angenayo i-nuclease okanye i-TE Buffer, ukuba kuyimfuneko.
4. Umthamo ocetyiswayo weesampuli ezahlukeneyo:
| Isampulu | Umthamo omnye 50 μL ukusabela | Ubuninzi umlinganiselo |
| Igazi elipheleleyo le-anticoagulated | 2.5 μL | 5% |
| I-Plasma | 15 μL | 30% |
| ISerum | 10 μL | 20% |
| Ukuswabha komqala | 10 μL | 20% |
| Amathe | 10 μL | 20% |
Ulawulo lwemeko
1. Ukubona umsebenzi: ubuntununtunu, ukuchaneka kunye nokuphindaphinda kwe-qPCR.
2. Akukho msebenzi we-nuclease exogenous: akukho endonuclease exogenous kunye exonuclease ungcoliseko.
Amanqaku eMveliso
1. Le mveliso isebenzisa uhlobo lwenoveli ye-polymerase ye-DNA eshushu, enokuthi isebenze kwi-1-5 imizuzu. Ekubeni i-buffer yayo ye-reaction iye yaphuculwa, ifaneleke ngakumbi kwi-PCR ephindwe kabini okanye emininzi ye-fluorescence quantitative usebenzisa indlela ye-probe.
2. Ukuba ixabiso le-Rn lokwandiswa kwe-PCR liphantsi kakhulu okanye i-amplification ivinjelwe ngokucacileyo, ukunciphisa inani lesampuli, ukwandisa umthamo wokuphendula okanye ukuhlanjululwa kwangaphambili kwesampulu kunokuphucula iziphumo.
3. Ukuqokelela kwegazi, amathe, umchamo, umqala we-swab, njl. kufuneka kulandele iimfuno zekhrayitheriya yeklinikhi, kwaye isampulu entsha ingasetyenziselwa ukuthintela ukuthotywa kwe-nucleic acid.
4. Ekubeni i-amplicons ezahlukeneyo zineempumelelo ezahlukeneyo zokusetyenziswa kwi-dUTP kunye novelwano kwi-UNG, i-reagents kufuneka iphuculwe ukuba ukufumanisa ukuvakalelwa kunciphisa xa usebenzisa inkqubo ye-UNG.Nceda uqhagamshelane nathi ngenkxaso yobugcisa xa kuyimfuneko.
5. Ukuze ugweme ukukhulisa iimveliso ze-PCR phakathi kweempendulo zenyathelo elinye, indawo yovavanyo olunikezelweyo kunye ne-pipette iyafuneka ukukhulisa.Sebenza ngeeglavu kwaye utshintshe rhoqo kwaye ungavuli ityhubhu yokusabela emva kokukhulisa i-PCR.
