Universal SYBR GREEN qPCR Premix (Blue)
Katsi Nhamba: HCB5041B
Universal Blue qPCR Master Mix (Dye Yakavakirwa) ndeye pre-mhinduro ye2 × chaiyo-nguva yakawanda PCR amplification inoratidzwa nehupamhi hwekunzwa uye hunhu, ibhuruu muruvara, uye ine mhedzisiro yemuenzaniso wekuwedzera kutsvaga.Iyo yakakosha chikamu Taq DNA polymerase inoshandisa antibody inopisa kutanga kuvharira kusiri-chaiyo amplification nekuda kweprimer annealing panguva yekugadzirira sampuli.Panguva imwecheteyo, fomula rinowedzera zvinhu zvinosimudzira kuvandudza kugona kwePCR kuita uye kuenzanisa kukwidziridzwa kwemajini ane akasiyana GC zvirimo (30 ~ 70%), kuitira kuti huwandu hwePCR huwane hukama hwakanaka hwemutsara muhuwandu hwakawanda. dunhu.Ichi chigadzirwa chine yakakosha ROX Passive Reference Dye, inoshanda kune akawanda qPCR zviridzwa.Izvo hazvifanirwe kugadzirisa kuwanda kweROX pane zvakasiyana zviridzwa.Izvo zvinongodiwa kuwedzera maprimers uye matemplate kugadzirira maitiro sisitimu yekusimudzira.
Zvikamu
Universal Blue qPCR Master Mix
Kuchengetedza mamiriro
Chigadzirwa chacho chinotumirwa nema ice packs uye chinogona kuchengetwa pa -25 ℃ ~ -15 ℃ kwemwedzi ye18.Izvo zvinodikanwa kudzivirira yakasimba mwenje irradiation paunenge uchichengeta kana kugadzirira reaction system.
Tsanangudzo
| Concentration | 2× |
| Nzira yekuona | SYBR |
| PCR nzira | qPCR |
| Polymerase | Taq DNA polymerase |
| Rudzi rwemuenzaniso | DNA |
| Zvishandiso zvekushandisa | Mazhinji qPCR zviridzwa |
| Chigadzirwa mhando | SYBR premix yeiyo chaiyo-nguva fluorescence kuwanda PCR |
| Nyorera ku (kunyorera) | Gene Expression |
Mirayiridzo
1.Reaction System
| Zvikamu | Ukuru (μL) | Ukuru (μL) | Final Concentration |
| Yese SYBR GREEN qPCR Premix | 25 | 10 | 1× |
| Pamberi Pekutanga (10μmol/L) | 1 | 0.4 | 0.2μmol/L |
| Reverse Primer (10μmol/L) | 1 | 0.4 | 0.2μmol/L |
| DNA | X | X | |
| ddH2O | kusvika ku50 | kusvika ku20 | - |
[Cherechedza]: Sanganisa zvakakwana usati washandisa kudzivirira mabhuru akawandisa kubva mukuzunungusa zvine simba.
a) Primer concentration: Yekupedzisira primer concentration ndeye 0.2μmol/L, uye inogonawo kugadziriswa pakati pe0.1 uye 1.0μmol/L sezvakakodzera.
b) Template concentration: Kana template isina kunyorwa cDNA stock solution, vhoriyamu inoshandiswa haifanire kudarika 1/10 yehuwandu hwehuwandu hwekuita kweqPCR.
c) Template dilution: Zvinokurudzirwa kudzikisa cDNA stock solution ne5-10 nguva.Iyo yakakwana huwandu hwetemplate yakawedzerwa iri nani kana iyo Ct kukosha yakawanikwa nekukudza iri 20-30 mitsetse.
d) Reaction system: Zvinokurudzirwa kushandisa 20μL kana 50μL kuve nechokwadi chekushanda uye kudzokorora kwechinangwa chekusimudzira gene.
e) Kugadzirira kweSisitimu: Ndokumbira ugadzirire mubhenji rakachena uye shandisa matipi uye maitiro machubhu pasina masara nuclease;zvinokurudzirwa kushandisa mazano ane filter cartridges.Dzivisa kusvibiswa kwemuchinjikwa uye kusvibiswa kweaerosol.
2.Reaction program
Standard Program
| Cycle step | Temp. | Nguva | Cycles |
| Initial denaturation | 95℃ | 2 min | 1 |
| Denaturation | 95℃ | 10 sec | 40 |
| Kuwedzera/Kuwedzera | 60 ℃ | 30 sec★ | |
| Melting curve stage | Zviridzwa Defaults | 1 | |
Quick Program
| Cycle step | Temp. | Nguva | Cycles |
| Initial denaturation | 95℃ | 30 sec | 1 |
| Denaturation | 95℃ | 3 sec | 40 |
| Kuwedzera/Kuwedzera | 60 ℃ | 20 sec★ | |
| Melting curve stage | Zviridzwa Defaults | 1 | |
[Cherechedza]: Chirongwa chekukurumidza chakakodzera kuwanda kwemajini, uye mapurogiramu akajairwa anogona kuyedzwa kune akaomesesa echipiri chimiro majini.
a) Anealing tembiricha uye nguva: Ndokumbira gadzirisa zvinoenderana nehurefu hweprimer uye chinangwa chejini.
b) Fluorescence chiratidzo chekutora (★): Ndokumbira isa maitiro ekuyedza zvinoenderana nezvinodiwa mumirairo yekushandisa chiridzwa.
c) Kunyunguduka curve: Iyo chiridzwa default chirongwa chinogona kushandiswa zvakajairika.
3. Mhedzisiro Ongororo
Zvishoma zvezvitatu zvebhayoloji zvaidiwa pakuedza kwehuwandu.Mushure mekuita, iyo amplification curve uye kunyunguduka curve inoda kusimbiswa.
3.1 Amplification curve:
Iyo yakajairwa amplification curve ndeye S-yakaumbwa.Quantitative analysis inonyanya kujeka apo kukosha kweCt kunowira pakati pe20 ne30. Kana Ct inokosha iri pasi pe10, zvakakosha kuderedza template uye kuita muedzo zvakare.Kana iyo Ct kukosha iri pakati pe30-35, zvinodikanwa kuwedzera template yekumisikidza kana kuwanda kweiyo reaction system, kuitira kuti uvandudze kugona kweamplification uye kuve nechokwadi chechokwadi chekuongorora mhedzisiro.Kana kukosha kweCt kwakakura kupfuura makumi matatu neshanu, mibairo yekuyedzwa haigone kuongorora huwandu hwekutaura kwejini, asi inogona kushandiswa kuongororwa kwemhando.
3.2 Kunyunguduka curve:
Iyo imwe chete yepamusoro yekunyunguduka curve inoratidza kuti maitiro ekuita zvakanaka uye kuongororwa kwehuwandu kunogona kuitwa;kana iyo yakanyunguduka curve inoratidza kaviri kana kuwanda kwepamusoro, kuongororwa kwehuwandu hakugone kuitwa.Iko kunyunguduka kunoratidza mapeaks maviri, uye zvinofanirwa kutonga kuti iyo isiri-yakananga peak iri primer dimer kana isiri-specific amplification neDNA agarose gel electrophoresis.Kana iri primer dimer, inokurudzirwa kudzikisa iyo primer concentration kana redesign maprimers ane yakakwira amplification kunyatsoita.Kana isiri-chaiyo amplification, ndokumbirawo uwedzere tembiricha yekumisa, kana gadziridza maprimers nehunyanzvi.
Notes
Ndokumbira upfeke PPE inodiwa, jasi rakadaro uye magurovhosi, kuve nechokwadi chehutano hwako uye kuchengetedzeka!
Ichi chigadzirwa ndechekutsvaga kushandiswa CHETE!
