5 × Neoscript Fast RT-qPCR Premix plus-UNG
Inombolo yekati: HCB5142A
I-Neoscript Fast RT Premix-UNG (Probe qRT-PCR) ngumxube ozinzile we-probe-based based mix olungele ukukhutshelwa umva kwenyathelo elinye kunye ne-PCR yobungakanani (qRT-PCR).Ixhasa ukuxuba kwangaphambili kwee-primers kunye ne-probes kwaye uhlale uzinzile emva kokugcinwa kwexesha elide kwiqondo lokushisa eliphantsi.Isampulu eza kuvavanywa inokongezwa ngokuthe ngqo xa usebenzisa, ngaphandle kokuvula ityhubhu eyongezelelweyo / ukusebenza kombhobho.Le mveliso ibonelela ngamacandelo, umz. i-polymerase ye-DNA eshushu, i-M-MLV, i-heat-labile uracil DNA glycosylase (TS-UNG), i-RNase Inhibitor, i-MgCl2, ii-dNTPs (kunye ne-dUTP endaweni ye-dTTP), kunye nezinzisi.Nge-genetically modified quick amplification reverse transcriptase kunye ne-DNA polymerase, kunokwenzeka ukugqiba i-PCR amplification ngaphakathi kwemizuzu engama-20-40.Le reagent isebenzisa isithinteli esikhethekileyo se-qPCR kunye ne-enzymes exubeneyo ye-anti-inhibitory amplification enzyme kunye ne-UNG enzyme.Ke ngoko, inokufumana ulwandiso olulungileyo lwejini ekujoliswe kuyo kwaye ithintele ukukhulisa ubuxoki okubangelwa yintsalela yePCR kunye nongcoliseko lwe-aerosol.Esi sixhobo siyahambelana nezixhobo ezininzi ze-PCR ze-fluorescence ezivela kubavelisi abafana ne-Applied Biosystems, i-Eppendorf, i-Bio-Rad kunye ne-Roche.
Icandelo
1.25×Neoscript Fast RTase/UNG Mix
2.I-5×Neoscript ekhawulezayo ye-RT 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.
ULungiselelo lweNkqubo ye-QRT-PCR yeReaction
| Amacandelo | 25μLInkqubo | 50μLInkqubo | Ukugxininiswa kokugqibela |
| I-5×Neoscript ekhawulezayo ye-RT Premix Buffer (dUTP) | 5μL | 10μL | 1× |
| 25×Neoscript Fast RTase/UNG Mix | 1μL | 2μL | 1× |
| 25×Primer-Probe Mixa | 1μL | 2μL | 1× |
| Ithempleyithi ye-RNAb | - | - | - |
| ddH2O | Ukuya kuthi ga kwi-25μL | Ukuya kuthi ga kwi-50μL | - |
1) a.Uxinzelelo lokugqibela lwe-primer ludla ngokuba ngu-0.2μM.Ukufumana iziphumo ezingcono, ugxininiso lwe-primer lunokuphuculwa phakathi koluhlu lwe-0.2-1μM.Ngokuqhelekileyo, uxinaniso lweprobe lunokuphuculwa phakathi koluhlu lwe-0.1-0.3μM.
2) b.Xa usebenzisa iNkqubo ye-PCR ekhawulezayo, ukunyusa ukuxinwa kwee-primers kunye neeprobes kunokubangela iziphumo ezingcono zokukhulisa, kwaye umlinganiselo wabo kufuneka uphuculwe ngokufanelekileyo.
I-3) Iintlobo ezahlukeneyo zeesampuli ziqulethe iintlobo ezahlukeneyo kunye nomxholo we-inhibitor kunye nekopi yekopi ye-gene ekujoliswe kuyo.Umthamo wesampuli kufuneka uqwalaselwe yimeko yangempela.Yenza i-dilution yesampuli ngamanzi angenayo i-nuclease okanye i-TE Buffer, ukuba kuyimfuneko.
Impendulo Ciimeko
| Inkqubo ye-PCR rhoqo | Inkqubo yePCR ekhawulezayo | ||||||
| Inkqubo | Temp. | Ixesha | Umjikelo | Inkqubo | Temp. | Ixesha | Umjikelo |
| Reverse Transcription | 50℃ | 10-20mins | 1 | Reverse Transcription | 50℃ | 5 imiz | 1 |
| Ipolymerase Ukuqaliswa | 95℃ | 1-5mins | 1 | Ipolymerase Ukuqaliswa | 95℃ | 30s | 1 |
| I-Denaturation | 95℃ | 10-20s | 40-50 | I-Denaturation | 95℃ | 1-3s | 40-50 |
| Ukuhlaziya kwaye Ulwandiso | 56-64℃ | 20-60s | Ukuhlaziya kwaye Ulwandiso | 56-64℃ | 3-20s | ||
Ulawulo lwemeko
1.Ukubona umsebenzi: ubuntununtunu, ukuchaneka kunye nokuphindaphinda kwe-qPCR.
2.Akukho msebenzi we-nuclease exogenous: akukho endonuclease exogenous kunye exonuclease ungcoliseko.
Amanqaku
1.Isantya sokukhulisa i-polymerase ye-DNA ekhawulezayo ayikho ngaphantsi kwe-1kb/10s.Izixhobo ezahlukeneyo zePCR zinesantya esahlukileyo sokufudumeza kunye nesantya sokupholisa, iindlela zokulawula ubushushu kunye ne-thermal conductivity, kwaye ngaloo ndlela ukulungelelaniswa kwe-primer / probe yakho yoxinaniso kunye nendlela yokuqhuba ngokudibanisa nesixhobo sakho esikhawulezayo sePCR kubalulekile.
2.Le mveliso yenza ukusetyenziswa okubanzi, kwaye ifanelekile kwi-high-sensitivity ye-molecular diagnosis.Indlela ye-PCR enamanyathelo amathathu iyacetyiswa kwii-primers ezinobushushu obuphantsi be-anneal okanye ukukhulisa amaqhekeza amade ngaphezulu kwe-200 bp.
3.Ekubeni i-amplicon ezahlukeneyo zineempumelelo ezahlukeneyo zokusetyenziswa kwe-dUTP kunye novakalelo olwahlukileyo kwi-UNG, ii-reagents kufuneka ziphuculwe ukuba uvakalelo lokubona luyancipha xa usebenzisa inkqubo ye-UNG.Nceda uqhagamshelane nathi ngenkxaso yobugcisa xa kuyimfuneko.
4.Ukuphepha ukukhulisa iimveliso ze-PCR, indawo yovavanyo olunikezelweyo kunye ne-pipette iyafuneka ukukhulisa.Sebenza ngeeglavu kwaye utshintshe rhoqo kwaye ungavuli ityhubhu ye-PCR emva kokukhulisa.
