Universal SYBR GREEN qPCR Premix (Putsoa)
Nomoro ea katse: HCB5041B
Universal Blue qPCR Master Mix (Dye Based) ke tharollo ea pele bakeng sa 2 × ea sebele ea nako ea sebele ea palo ea PCR e khetholloang ka kutloisiso e phahameng le ho khetheha, e putsoa ka 'mala,' me e na le phello ea mohlala oa ho kenyelletsa mohlala.Karolo ea mantlha ea Taq DNA polymerase e sebelisa antibody e chesang ho qala ho thibela ka mokhoa o hlakileng ho holisa ho sa totobalang ka lebaka la "primer annealing" nakong ea tokiso ea sampole.Ka nako e ts'oanang, foromo e eketsa lintlha tse khothalletsang ho ntlafatsa katleho ea karabelo ea PCR le ho lekanya ho holisoa ha liphatsa tsa lefutso tse nang le likahare tse fapaneng tsa GC (30 ~ 70%), e le hore palo ea PCR e ka fumana kamano e ntle ea linear ka bongata. sebaka.Sehlahisoa sena se na le ROX Passive Reference Dye e khethehileng, e sebetsang ho lisebelisoa tse ngata tsa qPCR.Ha ho hlokahale ho fetola khatello ea ROX ho lisebelisoa tse fapaneng.Hoa hlokahala feela ho kenyelletsa li-primers le litempele ho lokisa sistimi ea karabelo bakeng sa ho holisa.
Likaroloana
Universal Blue qPCR Master Mix
Maemo a polokelo
Sehlahisoa se romelloa ka lipakete tsa leqhoa 'me se ka bolokoa ho -25 ℃ ~ -15 ℃ bakeng sa likhoeli tsa 18.Hoa hlokahala ho qoba khanya e matla ea khanya ha u boloka kapa u lokisetsa mokhoa oa ho arabela.
Tlhaloso
| Ho tsepamisa maikutlo | 2× |
| Mokhoa oa ho lemoha | SYBR |
| Mokhoa oa ho sebelisa PCR | qPCR |
| Polymerase | Taq DNA polymerase |
| Mofuta oa mohlala | DNA |
| Lisebelisoa tsa kopo | Lisebelisoa tse ngata tsa qPCR |
| Mofuta oa sehlahisoa | SYBR premix bakeng sa nako ea nnete ea fluorescence quantitative PCR |
| Etsa kopo ho (ts'ebeliso) | Tlhaloso ea Gene |
Litaelo
1.Reaction System
| Likaroloana | Bolumo(μL) | Bolumo(μL) | Boikutlo ba ho Qetela |
| Universal SYBR GREEN qPCR Premix | 25 | 10 | 1× |
| Primer ea pele (10μmol/L) | 1 | 0.4 | 0.2μmol/L |
| Primer e Fetolelang (10μmol/L) | 1 | 0.4 | 0.2μmol/L |
| DNA | X | X | |
| ddH2O | ho fihlela ho 50 | ho fihlela ho 20 | - |
[Tlhokomeliso]: Kopanya hantle pele o sebelisoa ho qoba li-bubble tse feteletseng ho sisinyeha ho matla.
a) Khatiso ea pele: Khatiso ea ho qetela ea primer ke 0.2μmol / L, 'me e ka boela ea fetoloa pakeng tsa 0.1 le 1.0μmol / L ha ho loketse.
b) Khatiso ea template: Haeba thempleite e sa lokisoe ka tharollo ea stock ea cDNA, molumo o sebelisitsoeng ha oa lokela ho feta 1/10 ea kakaretso ea karabelo ea qPCR.
c) Ho hlapolla ha template: Ho khothalletsoa ho hlapolla tharollo ea setoko sa cDNA ka makhetlo a 5-10.Palo e nepahetseng ea template e kenyellelitsoeng e betere ha boleng ba Ct bo fumanoeng ka ho holisa ke li-cycle tse 20-30.
d) Sistimi ea karabelo: Ho khothaletsoa ho sebelisa 20μL kapa 50μL ho netefatsa katleho le ho pheta-pheta ha tokiso ea liphatsa tsa lefutso.
e) Tokiso ea sistimi: Ka kopo itokisetse bencheng e hloekileng haholo 'me u sebelise malebela le li-tubes tsa karabelo ntle le masala a nuclease;ho kgothaletswa ho sebelisa malebela ka li-cartridges tsa filter.Qoba tšilafalo e fapaneng le tšilafalo ea aerosol.
2.Lenaneo la ho arabela
Lenaneo le Tloaelehileng
| Mohato oa potoloho | Temp. | Nako | Lipotoloho |
| Denaturation ea pele | 95℃ | 2 mets | 1 |
| Denaturation | 95℃ | 10 sec | 40 |
| Katoloso/ Katoloso | 60 ℃ | metsotsoana e 30★ | |
| Mokhahlelo oa ho qhibiliha | Mehato ea Liletsa | 1 | |
Lenaneo le Potlakileng
| Mohato oa potoloho | Temp. | Nako | Lipotoloho |
| Denaturation ea pele | 95℃ | 30 metsotsoana | 1 |
| Denaturation | 95℃ | 3 metsotsoana | 40 |
| Katoloso/ Katoloso | 60 ℃ | metsotsoana e 20★ | |
| Mokhahlelo oa ho qhibiliha | Mehato ea Liletsa | 1 | |
[Tlhokomeliso]: Lenaneo le potlakileng le loketse boholo ba liphatsa tsa lefutso, 'me mananeo a tloaelehileng a ka lekoa bakeng sa liphatsa tsa lefutso tse rarahaneng tsa bobeli.
a) Thempereichara le nako: Ka kopo fetola ho latela bolelele ba mofuta oa pele le mofuta oa sepheo.
b) Ho fumana lets'oao la Fluorescence (★): Ka kopo, seta mokhoa oa liteko ho latela litlhoko tsa litaelo tsa tšebeliso ea sesebelisoa.
c) Melting Curve: Lenaneo la kamehla la lisebelisoa le ka sebelisoa ka tloaelo.
3. Sephetho Analysis
Ho ne ho hlokahala bonyane likopi tse tharo tsa baeloji bakeng sa liteko tsa bongata.Kamora karabelo, lekhalo la amplification le curve e qhibilihang li hloka ho netefatsoa.
3.1 Khatiso ea curve:
Mokokotlo o tloaelehileng oa amplification o bōpehile ka S.Tlhahlobo ea palo e nepahetse haholo ha boleng ba Ct bo oela pakeng tsa 20 le 30. Haeba boleng ba Ct bo le ka tlase ho 10, hoa hlokahala ho hlapolla thempleite le ho etsa tlhahlobo hape.Ha boleng ba Ct bo le lipakeng tsa 30-35, hoa hlokahala ho eketsa mahloriso a template kapa boholo ba sistimi ea karabelo, e le ho ntlafatsa ts'ebetso ea amplification le ho netefatsa ho nepahala ha tlhahlobo ea sephetho.Ha boleng ba Ct bo le boholo ho feta 35, liphetho tsa liteko li ke ke tsa hlahloba ka bongata polelo ea lefutso, empa li ka sebelisoa bakeng sa tlhahlobo ea boleng.
3.2 Mokokotlo o qhibilihang:
Tlhōrō e le 'ngoe ea lekhalo la ho qhibiliha e bontša hore tlhaloso ea karabelo e ntle le tlhahlobo ea bongata e ka etsoa;haeba sekhahla sa ho qhibiliha se bontša litlhōrō tse peli kapa tse ngata, tlhahlobo ea bongata e ke ke ea etsoa.Mokokotlo o qhibilihang o bonts'a litlhōrō tse peli, 'me hoa hlokahala ho ahlola hore na sehlohlolo se sa lebelloang ke primer dimer kapa amplification e seng e khethehileng ka DNA agarose gel electrophoresis.Haeba e le primer dimer, ho kgothaletswa ho fokotsa mahloriso a primer kapa ho tsosolosa li-primers ka katleho e phahameng ea amplification.Haeba e sa phahamisoe ka mokhoa o sa khetheheng, ka kopo eketsa mocheso oa li-annealing, kapa u hlophise li-primer ka mokhoa o ikhethileng.
Lintlha
Ka kopo, roala PPE e hlokahalang, jase e joalo ea lab le liatlana, ho netefatsa bophelo bo botle le polokeho ea hau!
Sehlahisoa sena ke sa tšebeliso ea lipatlisiso FEELA!
