5 × Neoscript Fast RT-qPCR Premix plus-UNG
Nomoro ea katse: HCB5142A
Neoscript Fast RT Premix-UNG (Probe qRT-PCR) ke motsoako o tsitsitseng oa tube e le 'ngoe e thehiloeng ho probe e loketseng mohato o le mong oa ho ngola reverse le PCR ea bongata (qRT-PCR).E ts'ehetsa ho kopanya pele ho li-primers le probes le ho lula u tsitsitse ka mor'a ho boloka nako e telele mocheso o tlase.Mohlala o lokelang ho lekoa o ka eketsoa ka kotloloho ha o sebelisoa, ntle le ts'ebetso e eketsehileng ea ho bula li-tube / pipetting.Sehlahisoa sena se fana ka likarolo, mohlala, hot-start DNA polymerase, M-MLV, heat-labile uracil DNA glycosylase (TS-UNG), RNase Inhibitor, MgCl.2, dNTPs (e nang le dUTP sebakeng sa dTTP), le li-stabilizers.Ka liphatsa tsa lefutso tse fetotsoeng ka potlako reverse transcriptase le DNA polymerase, hoa khonahala ho phethela ntlafatso ea PCR nakong ea metsotso e 20-40.Sehlahisoa sena se sebelisa buffer e khethehileng bakeng sa qPCR e nang le li-enzyme tse tsoakiloeng tsa enzyme ea amplification e khahlanong le inhibitory le enzyme ea UNG.Ka hona, e ka fumana matlafatso e ntle ea liphatsa tsa lefutso tse shebiloeng le ho thibela ho holisoa ha bohata ho bakoang ke masala a PCR le tšilafalo ea aerosol.Sesebelisoa sena se tsamaisana le lisebelisoa tse ngata tsa PCR tsa fluorescence quantitative tse tsoang ho bahlahisi ba kang Applied Biosystems, Eppendorf, Bio-Rad le Roche.
Karolo
1.25×Neoscript Fast RTase/UNG Mix
2.5 × Neoscript Fast RT Premix Buffer (dUTP)
Maemo a polokelo
Likarolo tsohle li lokela ho bolokoa ho -20 ℃ bakeng sa polokelo ea nako e telele le 4 ℃ bakeng sa likhoeli tse 3.Ka kopo kopanya hantle ka mor'a ho qhibiliha le centrifuge pele u sebelisa.Qoba ho qhibiliha khafetsa.
qRT-PCR Reaction System Tokiso
| Likaroloana | 25μLTsamaiso | 50μLTsamaiso | Boikutlo ba ho Qetela |
| 5 × Neoscript Fast RT Premix Buffer (dUTP) | 5μL | 10μL | 1× |
| 25×Neoscript Fast RTase/UNG Mix | 1μL | 2μL | 1× |
| 25×Primer-Probe Mixa | 1μL | 2μL | 1× |
| Setšoantšo sa RNAb | - | - | - |
| ddH2O | Ho fihlela ho 25μL | Ho fihlela ho 50μL | - |
1) a.Mokhahlelo oa ho qetela oa primer hangata ke 0.2μM.Bakeng sa liphetho tse ntle, mahloriso a primer a ka ntlafatsoa ka har'a mefuta ea 0.2-1μM.Ka kakaretso, mahloriso a probe a ka ntlafatsoa ka har'a mefuta e fapaneng ea 0.1-0.3μM.
2) b.Ha u sebelisa Mokhoa o potlakileng oa PCR, ho eketsa mahloriso a li-primers le li-probes ho ka etsa hore ho be le liphello tse ntle tsa amplification, 'me karo-karolelano ea bona e lokela ho ntlafatsoa ka tsela e nepahetseng.
3) Mefuta e fapaneng ea lisampole e na le mefuta e fapaneng le likahare tsa inhibitor le palo ea kopi ea mofuta oa sepheo.Molumo oa mohlala o lokela ho nkoa ka boemo ba sebele.Etsa dilution ea sampole ka metsi a se nang nuclease kapa TE Buffer, ha ho hlokahala.
Maikutlo Clintlafatso
| Mokhoa o tloaelehileng oa PCR | Mokhoa o potlakileng oa PCR | ||||||
| Tsamaiso | Temp. | Nako | Baesekele | Tsamaiso | Temp. | Nako | Baesekele |
| Reverse Transcription | 50 ℃ | 10-20mins | 1 | Reverse Transcription | 50 ℃ | 5 mets | 1 |
| Polymerase Tshebetso | 95℃ | 1-5 mets | 1 | Polymerase Tshebetso | 95℃ | 30s | 1 |
| Denaturation | 95℃ | 10-20s | 40-50 | Denaturation | 95℃ | 1-3s | 40-50 |
| Annealing le Katoloso | 56-64 ℃ | 20-60s | Annealing le Katoloso | 56-64 ℃ | 3-20s | ||
Taolo ea Boleng
1.Ho lemoha ha mosebetsi: kutlo, ho hlaka le ho pheta-pheta ha qPCR.
2.Ha ho ts'ebetso ea "nuclease exogenous": ha ho na endonuclease ea kantle le tšilafalo ea exonuclease.
Lintlha
1.Sekhahla sa ho holisa DNA polymerase e potlakileng ha se ka tlase ho 1kb/10s.Lisebelisoa tse fapaneng tsa PCR li na le lebelo le fapaneng la ho futhumatsa le ho pholisa, mekhoa ea taolo ea mocheso le conductivity ea mocheso, ka hona ho bohlokoa ho ntlafatsa mohopolo oa hau oa pele / probe le mokhoa oa ho sebetsa hammoho le sesebelisoa sa hau sa PCR se potlakileng.
2.Sehlahisoa sena se sebetsa ka bongata, 'me se loketse tlhahlobo ea limolek'hule tse phahameng haholo.Mokhoa oa PCR oa mehato e meraro o khothaletsoa bakeng sa li-primers tse nang le mocheso o tlase oa annealing kapa bakeng sa ho holisa likhechana tse telele ho feta 200 bp.
3.Kaha li-amplicon tse fapaneng li na le ts'ebeliso e fapaneng ea ts'ebeliso ea dUTP le kutlo e fapaneng ho UNG, li-reagents li lokela ho ntlafatsoa haeba kutloisiso ea ho lemoha e fokotseha ha u sebelisa sistimi ea UNG.Ka kopo ikopanye le rona bakeng sa tšehetso ea tekheniki ha ho hlokahala.
4.Ho qoba ho holisoa ha lihlahisoa tsa PCR, sebaka sa liteko le pipette lia hlokahala bakeng sa ho holisa.Sebetsa ka liatlana 'me u fetole khafetsa' me u se ke ua bula tšupu ea PCR ka mor'a ho holisa.
