Universal SYBR GREEN qPCR Premix (Blue)
Inombolo yekati: HCB5041B
I-Universal Blue qPCR Master Mix (i-Dye Based) sisisombululo sangaphambili se-2 × real-time quantitative quantitative PCR ephawulwa bubuntununtunu obuphezulu kunye nokuchaneka, inombala oluhlaza okwesibhakabhaka, kwaye inesiphumo sokulandela isampulu yokongeza.Elona candelo le-Taq DNA polymerase lisebenzisa i-antibody eshushu ukuqala ukunqanda ngokufanelekileyo ukukhulisa okungangqalanga ngenxa yokufakwa kwe-primer ngexesha lolungiselelo lwesampulu.Kwangaxeshanye, ifomula yongeza izinto ezikhuthazayo zokuphucula ukusebenza kakuhle kokukhulisa ukusabela kwe-PCR kunye nokulinganisa ukunyuswa kofuzo kunye nemixholo eyahlukeneyo yeGC (30 ~ 70%), ukuze i-PCR yobungakanani ifumane ubudlelwane obuhle bomgca kubungakanani obubanzi. ummandla.Le mveliso iqulethe i-ROX Passive Reference Dye ekhethekileyo, esebenzayo kuninzi lwezixhobo ze-qPCR.Akuyomfuneko ukulungelelanisa ukuxinana kwe-ROX kwizixhobo ezahlukeneyo.Kuyimfuneko kuphela ukongeza iiprimers kunye neetemplates ukulungiselela inkqubo yokusabela yokukhulisa.
Amacandelo
Universal Blue qPCR Master Mix
Iimeko zokugcina
Imveliso ithunyelwa ngeepakethe ze-ice kwaye ingagcinwa kwi -25 ℃ ~ -15 ℃ kwiinyanga ze-18.Kuyimfuneko ukuphepha ukukhanya okunamandla xa ugcina okanye ulungiselela inkqubo yokusabela.
Inkcazo
| Ukugxininisa | 2× |
| Indlela yokubona | I-SYBR |
| Indlela yePCR | qPCR |
| Ipolymerase | Taq DNA polymerase |
| Uhlobo lwesampulu | DNA |
| Izixhobo zesicelo | Uninzi lwezixhobo ze-qPCR |
| Uhlobo lwemveliso | I-SYBR premix ye-real-time fluorescence quantitative PCR |
| Faka isicelo ku (isicelo) | Gene Expression |
Imiyalelo
1.Reaction System
| Amacandelo | Umthamo(μL) | Umthamo(μL) | Ukugxininiswa kokugqibela |
| Universal SYBR GREEN qPCR Premix | 25 | 10 | 1× |
| Phambili iPrimer (10μmol/L) | 1 | 0.4 | 0.2μmol/L |
| Reverse Primer (10μmol/L) | 1 | 0.4 | 0.2μmol/L |
| DNA | X | X | |
| ddH2O | ukuya kuthi ga kwi-50 | ukuya kuthi ga kwi-20 | - |
[Qaphela]: Xuba ngocoselelo phambi kokusetyenziswa ukunqanda amaqamza agqithisileyo ekungcangcazeleni ngamandla.
a) Ugxininiso lwe-Primer: I-primer concentration yokugqibela yi-0.2μmol / L, kwaye inokulungelelaniswa phakathi kwe-0.1 kunye ne-1.0μmol / L ngokufanelekileyo.
b) Ugxininiso lwetemplate: Ukuba ithempleyithi ayixutywanga kwisisombululo sesitokhwe secDNA, umthamo osetyenzisiweyo akufanele udlule kwi-1/10 yomthamo opheleleyo wempendulo ye-qPCR.
c) I-template dilution: Kucetyiswa ukuba uhlambulule isisombululo sesitokhwe se-cDNA ngamaxesha angama-5-10.Isixa esifanelekileyo setemplate esongeziweyo singcono xa ixabiso le-Ct elifunyenwe ngokukhuliswa yi-20-30 imijikelezo.
d) Inkqubo yokusabela: Kucetyiswa ukuba kusetyenziswe i-20μL okanye i-50μL ukuqinisekisa ukusebenza kunye nokuphindaphinda kwe-target gene amplification.
e) Ukulungiswa kwenkqubo: Nceda ulungiselele kwibhentshi ecocekileyo kwaye usebenzise iingcebiso kunye neetyhubhu zokusabela ngaphandle kwentsalela ye-nuclease;kucetyiswa ukuba usebenzise iingcebiso ngeekhatriji zokucoca.Kuphephe ungcoliseko olunqamlezayo kunye nokungcoliseka kwe-aerosol.
2.Inkqubo yokusabela
Inkqubo esemgangathweni
| Inyathelo lomjikelo | Temp. | Ixesha | Imijikelo |
| I-denaturation yokuqala | 95℃ | 2 imiz | 1 |
| I-Denaturation | 95℃ | 10 imizuzwana | 40 |
| Ukwandiswa/Ukwandiswa | 60℃ | 30 imizuzwana★ | |
| Ukunyibilika kwegophe inqanaba | Ukungagqibeki kwesixhobo | 1 | |
Inkqubo ekhawulezayo
| Inyathelo lomjikelo | Temp. | Ixesha | Imijikelo |
| I-denaturation yokuqala | 95℃ | 30 imizuzwana | 1 |
| I-Denaturation | 95℃ | 3 imizuzwana | 40 |
| Ukwandiswa/Ukwandiswa | 60℃ | 20 imizuzwana★ | |
| Ukunyibilika kwegophe inqanaba | Ukungagqibeki kwesixhobo | 1 | |
[Qaphela]: Inkqubo ekhawulezayo ilungele uninzi lwemfuza, kwaye iinkqubo ezisemgangathweni zinokuzanywa kuhlobo oluthile lolwakhiwo oluntsonkothileyo lwesibini.
a) Ubushushu be-Anealing kunye nexesha: Nceda ulungelelanise ngokobude be-primer kunye nejene ekujoliswe kuyo.
b) Ukufunyanwa komqondiso weFluorescence (★): Nceda usete inkqubo yovavanyo ngokweemfuno kwimiyalelo yokusetyenziswa kwesixhobo.
c) Ijika lokunyibilikisa: Inkqubo engagqibekanga yesixhobo ingasetyenziswa ngokuqhelekileyo.
3. Uhlalutyo lweziphumo
Ubuncinci bezinto eziphindaphindwayo zebhayoloji bezifuneka kwimifuniselo yobungakanani.Emva kokusabela, igophe lokukhulisa kunye negophe lokunyibilika kufuneka kuqinisekiswe.
3.1 Igophe lokwandisa:
Umgangatho wegophe lokukhulisa u-S.Uhlalutyo lwamanani luchanekile kakhulu xa ixabiso le-Ct liwela phakathi kwe-20 kunye ne-30. Ukuba ixabiso le-Ct lingaphantsi kwe-10, kuyimfuneko ukuhlambulula i-template kwaye uqhube uvavanyo kwakhona.Xa ixabiso le-Ct liphakathi kwe-30-35, kuyimfuneko ukwandisa i-template yoxinaniso okanye umthamo wenkqubo yokuphendula, ukwenzela ukuba kuphuculwe ukusebenza kakuhle kwe-amplification kunye nokuqinisekisa ukuchaneka kohlalutyo lwesiphumo.Xa ixabiso le-Ct likhulu kune-35, iziphumo zovavanyo azikwazi ukuhlalutya ubungakanani bokubonakaliswa kwejini, kodwa zingasetyenziselwa uhlalutyo lomgangatho.
3.2 Ukunyibilika igophe:
I-peak eyodwa ye-melting curve ibonisa ukuba i-reaction specificity ilungile kwaye uhlalutyo lobungakanani lunokwenziwa;ukuba ijika elinyibilikayo libonisa iincopho eziphindwe kabini okanye ezininzi, uhlalutyo lobungakanani alunakwenziwa.Ijika elinyibilikayo libonisa iincopho eziphindwe kabini, kwaye kuyimfuneko ukugweba ukuba i-non-target peak yi-primer dimer okanye i-amplification engacaciswanga yi-DNA agarose gel electrophoresis.Ukuba i-primer dimer, kucetyiswa ukuba kuncitshiswe i-primer concentration okanye i-primers ngokutsha i-primers kunye nokusebenza kakuhle kokukhulisa.Ukuba akusiyo i-amplification engangqalanga, nceda unyuse iqondo lokushisa le-annealing, okanye uhlengahlengise iiprimers ngokuthe ngqo.
Amanqaku
Nceda unxibe iPPE eyimfuneko, idyasi yelebhu kunye neeglavu, ukuqinisekisa impilo yakho kunye nokhuseleko!
Le mveliso isetyenziselwa uphando KUPHELA!
