Superstart qPCR Premix iyo UNG
Bisadda No: HCB5071E
Superstart qPCR Premix plus-UNG waa reajin khaas ah oo loogu talagalay Real Time falcelinta tayada iyo tirada PCR iyadoo la adeegsanayo ogaanshaha ku saleysan baaritaanka, oo si gaar ah loogu sameeyay hababka lyophilization.Waxay ka kooban tahay novel hot-start enzyme Hotstart Taq plus (DG), kaas oo leh waxqabadkiisa Taq enzyme oo lagu shaabadeeyey heerkulka qolka, si wax ku ool ah u xannibaya kordhinta aan gaarka ahayn ee ay keento curiyaha aan khaaska ahayn ee soo-jiidashada ama sameynta dimer-ka hooseysa xaaladaha heerkulka hooseeya, sidaas awgeed hagaajinta gaar ahaan falcelinta xoojinta.Reagent-kani waxa uu isticmaalaa kayd gaar ah oo qPCR ah oo la hagaajiyay iyo UNG/dUTP nidaamka ka hortagga faddaraynta si loo gaadho kuleyl degdeg ah, si weyn u wanaajinaya waxtarka iyo dareenka falcelinta qPCR.Waxay ku heli kartaa qaloocyada heerka wanaagsan ee tiro balaadhan oo ah aagag qiyaaseed waxayna si sax ah u fulisaa tirooyinka, si wax ku ool ah ayey uga hortagtaa cod-weynayn been abuur ah oo ay sababto hadhaaga alaabada PCR ama wasakhowga hawada.Reagent-kani waxa uu la jaan qaadayaa inta badan qalabka PCR ee tirooyinka fluorescent ee soo saarayaasha sida Applied Biosystems, Eppendorf, Bio-Rad iyo Roche iwm.
Halabuurka reagent
1. 5× HotstartPremix iyo UNG (Mg2+bilaash ah) (DG)
2. 250 mM MgCl2
3. 4× lyoprotectant (ikhtiyaar)
Xaaladaha Kaydinta
Kaydinta muddada-dheer ee -20 ℃;waxa lagu kaydin karaa 4℃ ilaa 3 biloodSi fiican isku qas ka hor isticmaalka iyoIska ilaali qaboojinta soo noqnoqda iyo dhalaalidda.
Habka Baaskiilka
| Habraaca | Heerkulka | Waqtiga | Wareegto |
| Dheefshiidka | 50℃ | 2 daq | 1 |
| Dhaqdhaqaaqa polymerase | 95℃ | 1 ~ 5 daqiiqo | 1 |
| Denature | 95℃ | 10-20 s | 40-50 |
| Burinta iyo Kordhinta | 56 ~ 64 ℃ | 20-60 s | 40-50 |
qPCR Reaction System Diyaarinta
|
Halabuurka |
25µL mugga |
50µL mugga |
U-fiirsashada kama dambaysta ah |
| 5× HotstartPremix iyo UNG(Mg2+bilaash ah) (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× |
| Qaabka DNA-da3 | —— | —— | —— |
| ddH2O | ilaa 25µL | ilaa 50µL | —— |
1. Isku-duubnaanta ugu dambeysa ee 0.2μM ee asaasiga ah waxay badanaa keentaa natiijooyin wanaagsan;marka waxqabadka falcelintu xumaato, hagaaji feejignaanta asaasiga ah inta u dhaxaysa 0.2-1μM haddii loo baahdo.Fiirinta baaritaanka waxaa sida caadiga ah lagu hagaajiyay inta u dhaxaysa 0.1-0.3μM iyada oo loo marayo tijaabooyin tartiib tartiib ah si loo helo isku darka ugu fiican.
2. Tirada koobiga ee hiddo-wadeyaasha bartilmaameedka ah ee ka kooban noocyada kala duwan ee hababyada way kala duwan yihiin;Haddii loo baahdo, qasitaan gradient waa la samayn karaa si loo go'aamiyo tirada ku darida template ugu fiican.
3. Nidaamkani wuxuu noqon karaa lyophilized;Marka macaamiishu isticmaalaan nidaamkan iyada oo aan shuruudaha qalajinta, 4 × lyoprotectant ayaa si xushmad leh loogu dari karaa; haddii ay jiraan badeecooyin qallalan oo loo baahan yahay, inta lagu jiro marxaladda dareeraha dareeraha ah ee ansixinta waxqabadka alaabta, waa in lagu daraa 4 × lyoprotectant si loo hubiyo in ay la socdaan qaybaha nidaamka lyophilized iyo saamaynta.
Marka nidaamka la isticmaalod qalajinta qaboojinta, u diyaari nidaamka as soo socda:
| Halabuurka | 25µL Nidaamka falcelinta |
| 5 ×HotstartPremix iyo UNG (Mg2+bilaash ah) (DG) | 5µL |
| 250mM MgCl2 | 0.45µL |
| 4× lyoprotectant | 6.25µL |
| 25×Primer-Probe Mix | 1µL |
| ddH2O | Ilaa 18 ~ 20µL |
* Haddii nidaamyada kale ee qaboojinta-qallajinta loo baahan yahay, fadlan si gaar ah ula tasho.
Habka Lyophilizationss
| Habraaca | Heerkulka | Waqtiga | Xaalad | Cadaadis |
| Horay u qaboojin | 4℃ | 30 daqiiqo | Qabo |
1 atm |
| -50 ℃ | 60 daqiiqo | Qaboojin | ||
| -50 ℃ | 180 daqiiqo | Qabo | ||
| Qalajinta aasaasiga ah | -30℃ | 60 daqiiqo | Kululaynta |
vacuum Ultimate |
| -30℃ | 70 daqiiqo | Qabo | ||
| Qallajinta sare | 25℃ | 60 daqiiqo | Kululaynta |
vacuum Ultimate |
| 25℃ | 300 daq | Qabo |
2. Habka lyophilization ee kor ku xusan waa tixraac kaliya.Noocyada kala duwan ee alaabta iyo qaboojiyaha-qallajiyaha kala duwan waxay leeyihiin qiyaaso kala duwan, markaa hagaajinta waxaa lagu samayn karaa si waafaqsan xaqiiqadaxaaladaha inta lagu jiro isticmaalka.
3. Hababka lyophilization ee kala duwan ayaa laga yaabaa inay ku habboon yihiin cabbirrada kala duwan ee lyophilizedAlaabooyinka, markaa ansaxinta tijaabada ku filan waa in la sameeyaa marka loo isticmaalo wax soo saarka ballaaran.
Tilmaamaha isticmaalka lyophilized budo
1. Si kooban u baro budada lyophilized;
2. Ku dar template nucleic acid budada lyophilized oo ku dar biyo ilaa 25µL;
3. Si fiican u qas adigoo centrifugation ah oo ku ordi mashiinka.
Xakamaynta tayada
1. Tijaabada shaqaynaysa: dareenka, gaar ahaaneed, soo saarista qPCR.
2. Wax dhaqdhaqaaq nuclease ah ma jiro, ma jirto faddarayn endo/exogenous ah.
Macluumaadka Farsamada:
1. Superstart qPCR Premix plus-UNG waxay isticmaashaa enzym-bilow kulul oo cusub kaasoo awood u siinaya inuu si degdeg ah u bilaabo 1 ~ 5 daqiiqo gudahood;iyada oo loo marayo qaabaynta kaydiyaha gaarka ah waxay ku habboon tahay falcelinta PCR ee tirada badan ee fluorescent.
2. Waxay leedahay gaar gaar ah taas oo si weyn u wanaajisaa dareenka ee fluorescence tirada PCR xadka ogaanshaha, samaynta qalooca amplification caadi, qiimaha fluorescence helay horumar cad at templates fiirsashada hoose, ku haboon sida sare dareenka fluorescence tirada PCR reagents.
3. Qalabyada aasaasiga ah ee leh heerkulka xuubka hoose ama ka badan 200bp jajab, habka 3-tallaabo ayaa lagula talinayaa.
4. Waxtarka ka faa'iidaysiga dUTP iyo dareenka UNG enzyme waxay ku kala duwan yihiin hiddo-wadaha bartilmaameedka ah, sidaas darteed haddii la isticmaalo nidaamka UNG waxay keenaysaa hoos u dhaca dareenka ogaanshaha, habka falcelinta waa in la hagaajiyaa oo la hagaajiyaa.Haddii taageero farsamo loo baahan yahay fadlan la xidhiidh shirkadeena.
5. Isticmaal aagagga gaarka ah iyo tuubooyinka ka hor iyo ka dib cod-weyneysiinta, xidho galoofyada xilliga hawlgalka oo baddel iyaga;Ha furin tuubada falcelinta ka dib marka PCR la dhammeeyo si loo yareeyo faddaraynta shaybaarada alaabta PCR.
