Superstart qPCR Premix plus-UNG
Nomoro ea katse: HCB5071E
Superstart qPCR Premix plus-UNG ke sesebelisoa se ikhethileng se etselitsoeng karabelo ea boleng le bongata ba nako ea 'nete ea PCR se sebelisa tlhahlobo e thehiloeng ho probe, e ntlafalitsoeng ka ho khetheha bakeng sa lits'ebetso tsa lyophilization.E na le enzyme ea "hot-start" ea "Hotstart Taq plus" (DG), e nang le ts'ebetso ea eona ea enzyme ea Taq e tiisitsoeng mochesong oa kamore, e thibela ka mokhoa o hlakileng matlafatso e sa tobang e bakoang ke "primer non-specific annealing" kapa "primer dimer formation" tlas'a maemo a tlase a mocheso, kahoo e ntlafatsa. boikgethelo ba boitsoaro ba amplification.Sehlahisoa sena se sebelisa "buffer" e ntlafalitsoeng ea qPCR le sistimi e thibelang tšilafalo ea UNG/dUTP ho fihlella ho qala ho chesa ka potlako, ho ntlafatsa haholo ts'ebetso le kutlo ea karabelo ea qPCR.E ka fumana li-curve tse ntle tse tloaelehileng libakeng tse ngata tse fapaneng tsa quantitation le ho etsa palo e nepahetseng, ho thibela ka katleho matlafatso e fosahetseng e bakoang ke lihlahisoa tse setseng tsa PCR kapa tšilafalo ea aerosol.Sehlahisoa sena se tsamaisana le lisebelisoa tse ngata tsa PCR tsa fluorescent tse tsoang ho bahlahisi ba kang Applied Biosystems, Eppendorf, Bio- Rad le Roche joalo-joalo, 'me e bonts'a botsitso bo botle ka sebopeho sa lyophilized.
Sebopeho sa reagent
1. 5×HotstartPremix plus-UNG (Mg2+mahala) (DG)
2. 250 limilimithara MgCl2
3. 4×lyoprotectant (boikhethelo)
Maemo a polokelo
Ho boloka nako e telele ho -20 ℃;e ka bolokoa ho 4 ℃ ho fihlela likhoeli tse 3.Kopanya hantle pele o sebelisoa leqoba ho hatsela kgafetsa le ho qhibidiha.
Protocol ea Libaesekele
| Tsamaiso | Temp. | Nako | Baesekele |
| Tšilo ea lijo | 50 ℃ | 2 mets | 1 |
| Polymerase activation | 95℃ | 1 ~ 5 mets | 1 |
| Denature | 95℃ | 10~20 s | 40-50 |
| Anealing le Katoloso | 56 ~ 64 ℃ | 20~60 s | 40-50 |
qPCR Liquid Reaction System Tokisetso
|
Sebopeho |
25µL bophahamo ba modumo |
Bophahamo ba 50µL |
Boikutlo ba ho Qetela |
| 5×HotstartPremix hammoho le-UNG(Mg2+mahala) (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× |
| DNA ea Setšoantšo3 | —- | —- | —- |
| ddH2O | Ho isa ho 25µL | Ho fihlela ho 50µL | —- |
1. Khatiso ea ho qetela ea 0.2μM bakeng sa li-primers hangata e fana ka liphello tse ntle;ha ts'ebetso ea karabelo e fokola, fetola mohopolo oa primer ka har'a mefuta e fapaneng ea 0.2-1μM kamoo ho hlokahalang.Tlhokomelo ea probe hangata e ntlafatsoa ka har'a mefuta e fapaneng ea 0.1-0.3μM ka liteko tsa gradient ho fumana metsoako e nepahetseng.
2. Nomoro ea kopi ea liphatsa tsa lefutso tse shebiloeng tse fumanehang mefuteng e fapaneng ea litempele lia fapana;haeba ho hlokahala, ho hlapolloa ha gradient ho ka etsoa ho fumana palo e nepahetseng ea kenyelletso ea template.
3. Tsamaiso ena e ka ba lyophilized;ha bareki ba sebelisa sistimi ena ntle le litlhokahalo tsa ho omisa ka leqhoa, 4 ×lyoprotectant e ka eketsoa ka mokhoa o ikhethileng; haeba ho na le lihlahisoa tse omisitsoeng ka leqhoa tse hlokahalang, nakong ea netefatso ea ts'ebetso ea sehlahisoa sa li-reagents tsa mokelikeli, e tlameha ho eketsa 4 ×lyoprotectant ho netefatsa ho lumellana le likarolo tsa tsamaiso ea lyophilized. le liphello.
Ha sistimi e sebetsad bakeng sa ho omisa ka leqhoa, lokisetsa tsamaiso as tse latelang:
| Sebopeho | 25µL Reaction System |
| 5 ×HotstartPremix plus-UNG (Mg2+mahala) (DG) | 5µL |
| 250mM MgCl2 | 0.45µL |
| 4×Lyoprotectant | 6.25µL |
| 25×Primer-Probe Mix | 1µL |
| ddH2O | Ho fihlela ho 18-20µL |
* Haeba lits'ebetso tse ling tsa ho omisa lihatsetsi li hlokahala, ka kopo ikopanye ka thoko.
Mokhoa oa Lyophilizationss
| Tsamaiso | Temp. | Nako | Boemo | Khatello |
| Ho hatsetsa pele | 4℃ | 30 mets | Tšoara |
1 atm |
| -50 ℃ | 60 mets | Ho phodisa | ||
| -50 ℃ | 180 mets | Tšoara | ||
| Ho omisa ho Pele | -30 ℃ | 60 mets | Ho futhumatsa |
Ultimate Vacuum |
| -30 ℃ | 70 mets | Tšoara | ||
| Ho omisa ka bobeli | 25 ℃ | 60 mets | Ho futhumatsa |
Ultimate Vacuum |
| 25 ℃ | 300 mets | Tšoara |
2. Mokhoa o ka holimo oa lyophilization ke oa ho bua feela.Mefuta e fapaneng ea lihlahisoa le liomisa-mohatsetsi tse fapaneng li na le liparamente tse fapaneng, ka hona, ho ka etsoa liphetoho ho latela maemo a nnete.maemo nakong ya tshebediso.
3. Mekhoa e fapaneng ea lyophilization e ka ba e loketseng bakeng sa boholo bo fapaneng ba batch ea lyophilizedlihlahisoa, kahoo netefatso e lekaneng ea tlhahlobo e tlameha ho etsoa ha e sebelisoa bakeng sa tlhahiso e kholo.
Litaelo tsa ho sebelisa lyophilized phofo
1. Ka bokhutšoanyane centrifuge phofo ea lyophilized;
2. Kenya nucleic acid template ho phofo ea lyophilized 'me u kenye metsi ho fihlela ho 25µL;
3. Kopanya hantle ka centrifugation le matha ka mochine.
Taolo ea Boleng:
1. Teko ea ts'ebetso: kutloelo-bohloko, ho khetheha, ho ikatisa ha qPCR.
2. Ha ho ts'ebetso ea "nuclease exogenous", ha ho na endogenous endo/exonuclease tšilafalo.
Lintlha tsa Tekheniki:
1. Superstart qPCR Premix plus-UNG e sebelisa enzyme e ncha ea hot-start e nolofalletsang ho qala ho chesa ka potlako nakong ea metsotso e 1 ~ 5;ka mokhoa o khethehileng oa ho etsa buffer e loketse multiplex fluorescent quantitative PCR reactions.
2. E na le lintlha tse phahameng tse ntlafatsang haholo kutloisiso ea fluorescence quantitative PCR limit discovery, ho etsa amplification curves normalization, fluorescence value fumana ntlafatso e totobetseng ho li-templates tse tlaase tsa mahloriso, tse loketseng e le li-reagents tse phahameng tsa sensitivity fluorescence quantitative PCR.
3. Bakeng sa li-primers tse nang le mocheso o tlase oa annealing kapa nako e telele ho feta likhechana tsa 200bp, mokhoa oa 3-step o buelloa.
4. Ts'ebeliso e ntle ea dUTP le kutloisiso ho enzyme ea UNG e fapana bakeng sa liphatsa tsa lefutso tse fapaneng, kahoo haeba ho sebelisa tsamaiso ea UNG ho lebisa ho fokotseha ha kutloisiso ea ho lemoha, mokhoa oa ho itšoara o lokela ho lokisoa le ho ntlafatsoa.Haeba ho hlokahala tšehetso ea tekheniki ka kopo ikopanye le k'hamphani ea rona.
5. Sebelisa libaka tse inehetseng le li-pipette pele le ka mor'a ho hōlisa, roala liatlana nakong ea ts'ebetso 'me u li nke sebaka khafetsa;u se ke ua bula pompo ea karabelo ka mor'a ho phethoa ha PCR ho fokotsa tšilafalo ea lisampole ke lihlahisoa tsa PCR.
