
Degdega Ampli RT-qPCR Premix iyo UNG
Bisadda No: HCB5144E
Fast Ampli RT-qPCR Premix Plus-UNG waxaa si gaar ah loogu horumariyay hababka lyophilization, lagu dabaqay qiyaasta fluorescence (TaqMan probe) RNA ogaanshaha cod-weyneysiinta neocript reverse transcriptase iyo xoojinta degdegga ah ee DNA Polymerase oo la helay hiddo ahaan wax laga beddelay iyo baadhis taas oo dhamaystiri karta kordhinta PCR 20 gudahood. -40 daqiiqo.Reagent-kani waxa uu horumariyaa dulqaad sare oo ka hortag ah, kaas oo leh qoraal gadaal sare ah iyo hufnaanta PCR, oo ku habboon kor u qaadida dareenka-sare ee muunadaha RNA-fiirsiga hoose.Qalooca caadiga ah ee wanaagsan ayaa lagu heli karaa tiro balaadhan oo qiyaas ah, qiyaasidna waxaa loo samayn karaa si sax ah.Reagent-kani waxa uu isticmaalaa ensaymes isku-dhafan oo ah enzym-ka-hortagga-hor-istaagga xoojinta enzyme-ka iyo UNG enzyme, iyo sidoo kale hab-dhismeed la hagaajiyay oo ka kooban dUTP.Kaliya kuma gaari karto kordhinta wanaagsan ee hidde-sidaha bartilmaameedka ee shaybaarada ay ku jiraan xannibaadaha, laakiin sidoo kale waxay si wax ku ool ah uga hortagi kartaa cod-weyneyn been abuur ah oo ay keento wasakhowga PCR iyo aerosol.Reagent-kani waxa uu la jaan qaadayaa inta badan qalabyada PCR-ga ee fluorescent, sida Applied Biosystems, Eppendorf, Bio-Rad, Roche iyo wixii la mid ah.Reagent-kan waxa loo isticmaali karaa lyophilization si loo helo qaab lyophilized wanaagsan iyo xasilloonida alaabta.
Halabuurka reagent
1. 12.5×FastAmpli Qaybta Taq/UNG Mix (oo leh dNTPs) (DG)
2. 50× FastAmpli Qaybta RTase (DG)
3. 5 ×FastAmpliRT Buffer (DG)
4. 4 ×lyoprotectant (ikhtiyaar) (DG)
Xaaladaha Kaydinta
Waxa lagu kaydin karaa at -20℃ muddo dheer, at 4 ℃ ilaa 3 bilood.Si fiican isku qas ka hor isticmaalka oo iska ilaalibaraf-dhalaalid joogto ah.
Habka Baaskiilka
Talaabada |
Heerkulka | PCR joogta ah Habraaca | PCR degdeg ahHabraaca |
Wareegtada |
Muddada | Muddada | |||
GadaalQoraal | 50℃ | 15 daqiiqo | 5 daqiiqo | hayn |
PolymeraseFirfircoonida | 95℃ | 1-5 daqiiqo | 1-2 daqiiqo | hayn |
Denature | 95℃ | 10-20 s | 1-3 ilbiriqsi | 40-50 |
Baabi'inta/kordhinta | 56-64℃ | 20-60s | 3-20 ilbiriqsi |
Dareereyaasha RT-qPCRNidaamka ion
Halabuurka | 25µL Mugga | 50µL Mugga | Xoog-saarid |
5× FastAmpli RT Buffer | 5µL | 10µL | 1× |
12.5×FastAmpli Qaybta Taq/UNG Mix (oo leh dNTPs) | 2µL | 4µL | 1× |
50× FastAmpli Qaybta RTase | 0.5µL | 1µL | 1× |
4× lyoprotectant | 6.25µL | 12.5µL | 1× |
25×Primer-Probe Mix | 1µL | 2µL | 1× |
Template RNA | - | - | - |
ddH2O | ilaa 25µL | ilaa 50µL | 1× |
1. Nidaamkani waa nidaam lyophilization;Marka macaamiishu isticmaalaan nidaamkan iyada oo aan la helin shuruudaha lyophilization, 4 × lyoprotectant ayaa si xushmad leh loogu dari karaa;Haddii ay jiraan alaabo lyophilized loo baahan yahay, inta lagu guda jiro reagents dareere heerka ansaxinta waxqabadka alaabta, waa in ay ku darto 4 × lyoprotectant si loo hubiyo in joogto ah qaybaha nidaamka lyophilized iyo saamaynta.
2. Marka la isticmaalo nidaamka caadiga ah ee PCR, xooga saarista ugu dambeysa ee asaasiga ah badanaa waa 0.2μM.Si loo helo natiijooyin ka wanaagsan, xoogga saarista asaasiga ah waxaa lagu hagaajin karaa inta u dhaxaysa 0.2-1.0μM.U-fiirsashada baaritaanka waxaa lagu hagaajin karaa inta u dhexeysa 0.1-0.3μM.Tijaabooyin isugeyn xoog leh ayaa la sameyn karaa si loo helo isku dhafka ugu wanaagsan ee furayaasha iyo baarayaasha.
3. Markaad isticmaalayso habka kor u qaadida PCR ee degdega ah, natiijo ka fiican ayaa lagu gaadhi karaa iyadoo la kordhinayo feejignaanta asaasiga ah/baaritaannada, iyo sidoo kale hagaajinta boqolkiiba qoraha iyo baaritaanka.
4. Noocyada kala duwan waxay ka kooban yihiin tiro koobiyo kala duwan oo hiddo-wadaha bartilmaameedka ah.Kala-badhsi-jilicsan ayaa la samayn karaa si loo doorto qaddarka ku-darka qaab-dhismeedka ugu habboon, haddii loo baahdo.
Diyaarinta nidaamka Lyophilization
Halabuurka | 25µL falcelinta Nidaamka |
5× FastAmpli RT Buffer | 5µL |
12.5×FastAmpli Qaybta Taq/UNG Mix (oo leh dNTPs) (DG) | 2µL |
50×FastAmpli Qaybta RTase (DG) | 0.5µL |
4×Lyoprotectant | 6.25µL |
25×Primer-Probe Mix | 1µL |
ddH2O | Ilaa 18 ~ 20µL |
* Haddii nidaamyada kale ee lyophilization loo baahan yahay, fadlan si gaar ah ula tasho.
Lyophilization Process
Habraaca | Heerkulka | Waqtiga | Xaalad | Cadaadis |
Qabow | 4℃ | 30 daqiiqo | Qabo | 1 atm |
-50 ℃ | 60 daqiiqo | Qaboojin | ||
-50 ℃ | 180 daqiiqo | Qabo | ||
Qalajinta aasaasiga ah | -30℃ | 60 daqiiqo | Kululaynta | vacuum Ultimate |
-30℃ | 720 min | Qabo | ||
Qallajinta sare | 25℃ | 60 daqiiqo | Kululaynta | vacuum Ultimate |
25℃ | 300 daq | Qabo |
1. Habkan lyophilization waa habka qalajinta barafaynta ee goobta dhexdeeda ee habka falcelinta 25µL;Haddii ul qalajinta qaboojinta ama hababka kale ee qallajinta goobta dhexdeeda loo baahan yahay, fadlan si gaar ah u weydii.
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 iyadoo loo eegayo xaaladaha dhabta ah inta lagu jiro isticmaalka.
3. Nidaamyada lyophilization ee kala duwan ayaa laga yaabaa inay ku habboonaadaan cabbirrada kala duwan ee alaabta lyophilized, markaa waa in la sameeyaa imtixaan ku filan marka loo isticmaalo wax soo saarka ballaaran.
Tilmaamaha isticmaalka lyophilized budada
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 RT-qPCR.
2. Wax dhaqdhaqaaq nuclease ah ma jiro, ma jirto faddarayn endo/exogenous ah.
Macluumaadka Farsamada:
1. Heerka kordhinta polymerase DNA ee degdega ah kama yarayn 1kb/10s.Qalabyada PCR ee kala duwan waxay leeyihiin xawli kuleyl iyo qaboojin oo kala duwan, hababka xakamaynta heerkulka iyo dhaqdhaqaaqa kulaylka, sidaas awgeed hagaajinta diiradaada asaasiga ah iyo habka socodsiinta marka lagu daro qalabkaaga PCR ee degdega ah waa lama huraan.
2. Wakhtiga qoraalka dib u celinta ayaa la wanaajiyey iyadoo loo eegayo dhererka jajabka la kordhinayo.Jajabyada dheer, wakhtiga qoraalka qoraalka ah ayaa si habboon loo kordhin karaa.
3. Heerkulka fidinta balaadhinta waa in la hagaajiyaa iyadoo lagu salaynayo qiimaha Tm ee baaritaanka asaasiga ah iyo xaaladaha dhabta ah ee falcelinta.
4. Qalabyada aasaasiga ah ee leh heerkulka xuubka hoose ama ka badan 200 bp jajab, habka 3-tallaabo ayaa lagula talinayaa.
5. Fadlan isticmaal meelaha gaarka ah iyo pipettes ka hor iyo ka dib cod-weyneysiinta, xidho galoofyada oo si joogto ah u beddel;Ha furin tuubada falcelinta ka dib marka falcelinta PCR-ga la dhammeeyo si loo yareeyo faddaraynta shaybaarada alaabta PCR.
6. 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 nala soo xidhiidh.