Superstart qPCR Premix plus-UNG
Inombolo yekati: HCB5071E
I-Superstart qPCR Premix plus-UNG yi-reagent ekhethekileyo eyenzelwe ixesha lokwenyani le-PCR lomgangatho kunye neempendulo zobungakanani kusetyenziswa ubhaqo olusekwe kwi-probe, ephuhliswe ngokukodwa kwiinkqubo ze-lyophilization.Iqulethe inoveli i-enzyme eshushu-yokuqala iHotstart Taq plus (DG), enomsebenzi wayo we-enzyme ye-Taq etywinwe kwiqondo lobushushu begumbi, inqanda ngokufanelekileyo ukukhulisa okungangqalanga okubangelwa yi-primer non-specific annealing okanye i-primer dimer formation phantsi kweemeko zobushushu obuphantsi, ngaloo ndlela iphucula. ulwazi oluthile lwempendulo yokukhulisa.Esi sixhobo sisebenzisa i-qPCR ephuculweyo ye-buffer kunye ne-UNG/dUTP yokuchasana nokungcoliseka inkqubo ukuphumeza ukuqalisa okushushu okukhawulezileyo, kuphuculwe kakhulu ukusebenza kakuhle kunye novakalelo lokuphendula kwe-qPCR.Iyakwazi ukufumana iigophe ezisemgangathweni ezisemgangathweni kwiindawo ezininzi zokulinganisa kwaye ngokuchanekileyo wenze ubungakanani, ukuthintela ngokufanelekileyo ukunyuswa kobuxoki okubangelwa yimveliso ye-PCR eseleyo okanye ukungcoliswa kwe-aerosol.Le reagent iyahambelana nezixhobo ezininzi ze-PCR zobungakanani befluorescent ezivela kubavelisi abafana ne-Applied Biosystems, i-Eppendorf, i-Bio- Rad kunye ne-Roche njl., kwaye ibonisa uzinzo oluhle kwi- lyophilized form.
Ukuqulunqwa kwe-reagent
1. 5×HotstartPremix plus-UNG (Mg2+simahla) (DG)
2. 250 mM MgCl2
3. 4×lyoprotectant (uyazikhethela)
Iimeko zokuGcina
Ukugcinwa kwexesha elide kwi -20 ℃;ingagcinwa kwi-4℃ ukuya kwiinyanga ezi-3.Hlanganisa kakuhle ngaphambi kokusetyenziswa kunyekuphephe ukukhenkceza okuphindaphindiweyo kunye nokunyibilika.
Umgaqo weBhayisekile
| Inkqubo | Temp. | Ixesha | Umjikelo |
| Ukwetyisa | 50℃ | 2 imiz | 1 |
| Ukusebenza kwePolymerase | 95℃ | 1-5 imiz | 1 |
| Denature | 95℃ | 10-20 s | 40-50 |
| Ukwandiswa kunye noKwandiswa | 56 ~ 64℃ | 20-60 s | 40-50 |
qPCR Liquid Reaction Syisiqu Ukulungiselela
|
Ukuqamba |
25µL Umthamo |
50µL Umthamo |
Ukugxininiswa kokugqibela |
| 5×HotstartPremix plus-UNG(Mg2+simahla) (DG) | 5µL | 10µL | 1× |
| 250mM MgCl2 | 0.45µL | 0.9µL | 4.5 mm |
| 4× lyoprotectant1 | 6.25µL | 12.5µL | 1× |
| 25×Primer-Probe Mix2 | 1µL | 2µL | 1× |
| Isakhelo seDNA3 | —- | —- | —- |
| ddH2O | Ukuya kwi-25µL | Ukuya kwi-50µL | —- |
1. Ugxininiso lokugqibela lwe-0.2μM kwii-primers ludla ngokuvelisa iziphumo ezilungileyo;xa ukusebenza kwempendulo kungalunganga, hlengahlengisa ugxininiso lweprimer ngaphakathi koluhlu lwe-0.2-1μM njengoko kufuneka.Ukugxininiswa kweprobe ngokuqhelekileyo kulungiselelwe phakathi koluhlu lwe-0.1-0.3μM ngokusebenzisa imifuniselo yegradient ukufumana imidibaniso eyiyeyona.
2. Inani lekopi yeegenes ekujoliswe kuzo eziqulethwe kwiintlobo ngeentlobo zeethemplethi ziyahluka;ukuba kuyimfuneko, uhlambululo lwegradient lunokwenziwa ukumisela elona nani lifanelekileyo longezelelo lwetemplate.
3. Le nkqubo ingaba lyophilized;xa abathengi basebenzisa le nkqubo ngaphandle kweemfuno zokomisa umkhenkce, i-4 × lyoprotectant ingadityaniswa ngokukhethiweyo; ukuba kukho imveliso eyomisiweyo efunekayo, ngexesha le-reagents yolwelo lwenqanaba lokuqinisekisa ukusebenza kwemveliso, kufuneka yongeze i-4×lyoprotectant ukuqinisekisa ukuhambelana namacandelo enkqubo ye-lyophilized. kunye neziphumo.
Xa inkqubo isetyenziswad umkhenkce-womisa, lungiselela inkqubo as okulandelayo:
| Ukuqamba | 25µL Indlela yokusabela |
| 5 ×HotstartPremix plus-UNG (Mg2+simahla) (DG) | 5µL |
| 250mM MgCl2 | 0.45µL |
| 4× lyoprotectant | 6.25µL |
| 25×Primer-Probe Mix | 1µL |
| ddH2O | Ukuya kwi-18~20µL |
• Ukuba ezinye iinkqubo zokumisa umkhenkce ziyafuneka, nceda udibane ngokwahlukeneyo.
Inkqubo yeLyophilizationss
| Inkqubo | Temp. | Ixesha | Imeko | Uxinzelelo |
| Ukukhenkceza kwangaphambili | 4℃ | 30 imiz | Bamba |
1 atm |
| -50℃ | 60 imiz | Ukupholisa | ||
| -50℃ | 180 imiz | Bamba | ||
| Ukomisa okuPhambili | -30℃ | 60 imiz | Ukufudumeza |
Vacuum yokugqibela |
| -30℃ | 70 imiz | Bamba | ||
| Ukomisa okwesibini | 25℃ | 60 imiz | Ukufudumeza |
Vacuum yokugqibela |
| 25℃ | 300 imiz | Bamba |
2. Le nkqubo ye-lyophilization ingentla ibhekiselele kuphela.Iindidi ezahlukeneyo zeemveliso kunye nezomisi ezahlukeneyo zineeparamitha ezahlukeneyo, ke uhlengahlengiso lunokwenziwa ngokwenyani.iimeko ngexesha lokusetyenziswa.
3. Iinkqubo ezahlukeneyo ze-lyophilization zinokuthi zilungele ubungakanani bebhetshi ezahlukeneyo ze-lyophilizediimveliso, ngoko ke ukuqinisekiswa kovavanyo olwaneleyo kufuneka kwenziwe xa kusetyenziselwa imveliso enkulu.
Umyalelo wokusetyenziswa kwe-lyophilized umgubo
1. Ngokufutshane centrifuge umgubo lyophilized;
2. Yongeza itemplate ye-nucleic acid kumgubo we-lyophilized kwaye wongeze amanzi ukuya kwi-25µL;
3. Hlanganisa kakuhle nge-centrifugation kwaye usebenze kumatshini.
Ulawulo lwemeko:
1. Uvavanyo olusebenzayo: ubuntununtunu, ukuchaneka, ukuveliswa kwakhona kwe-qPCR.
2. Akukho msebenzi wangaphandle we-nuclease, akukho endogenous endo/exonuclease ungcoliseko.
Ulwazi lobuGcisa:
1. I-Superstart qPCR Premix plus-UNG isebenzisa inoveli eshushu-start enzyme eyenza ukuba kuqalise ubushushu obukhawulezayo kwimizuzu eyi-1 ~ 5;ngokusebenzisa ukwakheka okukhethekileyo kwesithinteli ilungele ukuphindaphindwa kwe-PCR yobungakanani befluorescent.
2. Inento ethile ephezulu ephucula kakhulu ubuntununtunu be-fluorescence quantitative PCR ukubhaqwa komda, ukwenza i-aplification curves normalization, ixabiso le-fluorescence lifumane uphuculo olucacileyo kwiitemplates zoxinaniso oluphantsi, olufaneleke njengobuntununtunu obuphezulu be-fluorescence quantitative reagents ye-PCR.
3. Kwii-primers ezinobushushu obuphantsi be-annealing okanye ubude kunamaqhekeza angama-200bp, indlela ye-3-step inconywa.
4. Ukusebenza kakuhle kokusetyenziswa kwe-dUTP kunye nobuntununtunu kwi-enzyme ye-UNG iyahluka kwiijene ezijoliswe kuzo ezahlukeneyo, ngoko ke ukuba ukusebenzisa inkqubo ye-UNG kukhokelela ekunciphiseni uvakalelo lokubona, inkqubo yokusabela kufuneka ilungiswe kwaye ilungiswe.Ukuba inkxaso yobugcisa iyafuneka nceda uqhagamshelane nenkampani yethu.
5. Sebenzisa iindawo ezinikezelweyo kunye neepipettes ngaphambi nangemva kokukhulisa, nxiba iiglavu ngexesha lokusebenza kwaye uzitshintshe rhoqo;musa ukuvula ityhubhu yokusabela emva kokugqitywa kwe-PCR ukunciphisa ukungcoliseka kweesampuli ngeemveliso ze-PCR.
