I-Universal SYBR GREEN qPCR Premix (Blue)
Inombolo yekati: HCB5041B
I-Universal Blue qPCR Master Mix (i-Dye Based) iyisixazululo sangaphambilini sokukhuliswa komthamo we-PCR wesikhathi sangempela esingu-2x esibonakala ngokuzwela okuphezulu nokucaciswa kwawo, siluhlaza okwesibhakabhaka ngombala, futhi sinomphumela wokulandela umkhondo wokwengezwa kwesampula.Ingxenye eyinhloko ye-Taq DNA polymerase isebenzisa isiqalo esishisayo se-antibody ukuze ivimbele ngempumelelo ukukhuliswa okungaqondile ngenxa yokufakwa kwe-primer ngesikhathi sokulungiselela isampula.Ngasikhathi sinye, ifomula yengeza izici ezikhuthazayo zokuthuthukisa ukusebenza kahle kokukhulisa ukusabela kwe-PCR nokulinganisa ukukhuliswa kwezakhi zofuzo ezinokuqukethwe okuhlukile kwe-GC (30 ~ 70%), ukuze i-PCR yobuningi ikwazi ukuthola ubudlelwano obuhle bomugqa ngenani elibanzi. isifunda.Lo mkhiqizo uqukethe i-ROX Passive Reference Dye ekhethekile, esebenza kumathuluzi amaningi we-qPCR.Akudingekile ukulungisa ukugcwala kwe-ROX kumathuluzi ahlukene.Kudingeka kuphela ukwengeza ama-primers namathempulethi ukuze ulungiselele uhlelo lokusabela lokukhulisa.
Izingxenye
I-Universal Blue qPCR Master Mix
Izimo zokugcina
Umkhiqizo uthunyelwa ngamaphakethe eqhwa futhi ungagcinwa ku -25 ℃~-15 ℃ izinyanga eziyi-18.Kuyadingeka ukugwema ukukhanya okunamandla lapho ugcina noma ulungiselela uhlelo lokusabela.
Ukucaciswa
| Ukugxila | 2× |
| Indlela yokuthola | I-SYBR |
| Indlela ye-PCR | qPCR |
| I-Polymerase | Taq DNA polymerase |
| Uhlobo lwesampula | I-DNA |
| Imishini yokufaka isicelo | Inani eliphakeme kakhulu lama-QPCR |
| Uhlobo lomkhiqizo | I-SYBR premix ye-real-time fluorescence quantitative PCR |
| Faka isicelo ku-(isicelo) | I-Gene Expression |
Iziyalezo
1.Isistimu yokusabela
| Izingxenye | Ivolumu(μL) | Ivolumu(μL) | Ukugxila Kokugcina |
| I-Universal SYBR GREEN qPCR Premix | 25 | 10 | 1× |
| I-Primer Phambili (10μmol/L) | 1 | 0.4 | 0.2μmol/L |
| I-Primer Reverse (10μmol/L) | 1 | 0.4 | 0.2μmol/L |
| I-DNA | X | X | |
| ddH2O | kufika ku-50 | kufika ku-20 | - |
[Qaphela]: Xuba kahle ngaphambi kokusetshenziswa ukuze ugweme amabhamuza amaningi ekunyakazeni okunamandla.
a) I-Primer concentration: I-primer concentration yokugcina ingu-0.2μmol/L, futhi ingabuye ilungiswe phakathi kuka-0.1 no-1.0μmol/L ngokufanelekile.
b) Ukugxiliswa kwesifanekiso: Uma isifanekiso siyisixazululo sesitoko se-cDNA esingaxutshiwe, umthamo osetshenzisiwe akufanele udlule u-1/10 wevolumu ephelele yokusabela kwe-qPCR.
c) Ukuhlanjululwa kwesifanekiso: Kunconywa ukuthi uhlambulule isisombululo sesitoko se-cDNA izikhathi ezi-5-10.Inani elilungile lesifanekiso esingezwe lingcono uma inani le-Ct elitholwe ngokukhuliswa liyimijikelezo engu-20-30.
d) Uhlelo lokusabela: Kunconywa ukusebenzisa u-20μL noma u-50μL ukuze kuqinisekiswe ukusebenza kahle nokuphindaphinda kokukhulisa isakhi sofuzo.
e) Ukulungiswa kwesistimu: Sicela ulungiselele ebhentshini elihlanzekile kakhulu futhi usebenzise amathiphu namashubhu okusabela ngaphandle kwezinsalela ze-nuclease;kunconywa ukusebenzisa amathiphu nge-cartridges yokuhlunga.Gwema ukungcola okuphambanayo kanye nokungcoliswa kwe-aerosol.
2.Uhlelo lokusabela
Uhlelo olujwayelekile
| Isinyathelo somjikelezo | Temp. | Isikhathi | Imijikelezo |
| I-denaturation yokuqala | 95℃ | 2 imiz | 1 |
| I-Denaturation | 95℃ | 10 isekhondi | 40 |
| I-Annealing/Extension | 60℃ | 30 isekhondi★ | |
| Isigaba sejika elincibilikayo | Okuzenzakalelayo Kwezinsimbi | 1 | |
Uhlelo Olusheshayo
| Isinyathelo somjikelezo | Temp. | Isikhathi | Imijikelezo |
| I-denaturation yokuqala | 95℃ | 30 isekhondi | 1 |
| I-Denaturation | 95℃ | 3 isekhondi | 40 |
| I-Annealing/Extension | 60℃ | 20 isekhondi★ | |
| Isigaba sejika elincibilikayo | Okuzenzakalelayo Kwezinsimbi | 1 | |
[Qaphela]: Uhlelo olusheshayo lufanele iningi lezakhi zofuzo, futhi izinhlelo ezijwayelekile zingazanywa kuzakhi zofuzo zesakhiwo sesibili eziyinkimbinkimbi.
a) Izinga lokushisa le-anneal kanye nesikhathi: Sicela ulungise ngokuya ngobude be-primer kanye nofuzo oluqondiwe.
b) Ukutholwa kwesiginali ye-Fluorescence (★): Sicela usethe inqubo yokuhlola ngokuya ngezidingo emiyalweni yokusebenzisa ithuluzi.
c) Ijika elincibilikayo: Uhlelo oluzenzakalelayo lwensimbi lungasetshenziswa ngokujwayelekile.
3. Ukuhlaziywa Kwemiphumela
Ubuncane bokuphindaphinda okuthathu kwebhayoloji bekudingeka ekuhlolweni komthamo.Ngemuva kokusabela, ijika le-amplification kanye nejika elincibilikayo lidinga ukuqinisekiswa.
3.1 Ijika lokukhulisa:
Ijika lokukhulisa elijwayelekile linomumo ongu-S.Ukuhlaziywa komthamo kunembe kakhulu lapho inani le-Ct liwela phakathi kuka-20 no-30. Uma inani le-Ct lingaphansi kuka-10, kuyadingeka ukuhlambulula isifanekiso bese uphinda wenze ukuhlola.Uma inani le-Ct liphakathi kuka-30-35, kuyadingeka ukwandisa ukugxiliswa kwesifanekiso noma umthamo wesistimu yokusabela, ukuze kuthuthukiswe ukusebenza kahle kokukhulisa nokuqinisekisa ukunemba kokuhlaziywa kwemiphumela.Uma inani le-Ct likhulu kuno-35, imiphumela yokuhlolwa ayikwazi ukuhlaziya ngokwesilinganiso isisho sofuzo, kodwa ingasetshenziselwa ukuhlaziya ikhwalithi.
3.2 Ijika elincibilikayo:
Ukuphakama okukodwa kwejika elincibilikayo kubonisa ukuthi ukusabela okucacile kuhle futhi ukuhlaziywa komthamo kungenziwa;uma ijika elincibilikayo libonisa ukuphakama okuphindwe kabili noma okuningi, ukuhlaziywa kobuningi akukwazi ukwenziwa.Ijika elincibilikayo libonisa ukuphakama okuphindwe kabili, futhi kuyadingeka ukwahlulela ukuthi isiqongo esingaqondile siyi-primer dimer noma ukukhulisa okungaqondile nge-DNA agarose gel electrophoresis.Uma kuyi-primer dimer, kunconywa ukuthi unciphise ukugxila kwe-primer noma uhlele kabusha ama-primers ngokusebenza kahle kwe-amplification ephezulu.Uma kungekona ukukhulisa okungaqondile, sicela ukhuphule izinga lokushisa le-annealing, noma udizayine kabusha ama-primer ngokucacisa.
Amanothi
Sicela ugqoke i-PPE edingekayo, ijazi lelebhu kanye namagilavu, ukuze uqinisekise impilo yakho nokuphepha!
Lo mkhiqizo owokusetshenziswa ucwaningo KUPHELA!
