2 × Sensi Direct Premix-UNG (Probe qPCR)
Mpempe nwamba: HCB5151A
Emebere SensiDirect Premix-UNG (Probe qPCR) iji rụọ PCR ozugbo site na nlele na-enweghị mmịpụta DNA ma ọ bụ nkwadebe nlele.Nke a reagent mejupụtara DNA polymerase na-ekpo ọkụ, uracil DNA glycosylase (UNG), RNase Inhibitor, MgCl.2, dNTPs (ya na dUTP kama dTTP), na stabilizers, maka PCR quantitative (qPCR).Nke a reagent preforms elu inhibitor ndidi, ya mere, ọ nwere ike tinye ozugbo na nchọpụta nke samples dị ka akpịrị swab, asu, mgbochi-coagulated ọbara dum, plasma, na serum na-enweghị DNA mmịpụta.Reagent na-eji nchekwa nke qPCR nwere enzymes agwakọta nke DNA polymerase mgbochi mgbochi yana UNG enzyme.Ya mere, ọ nwere ike nweta nkwalite dị mma nke mkpụrụ ndụ ihe nketa lekwasịrị anya n'omume nwere ihe mgbochi ma gbochie mgbasawanye nke ọma nke PCR na mmetọ ikuku.Nke a reagent dakọtara na ọtụtụ fluorescent quantitative ngwa PCR, dị ka Applied Biosystems, Eppendorf, Bio-Rad, Roche na na.
Ngwa
1. 50× SensiDirect Enzyme/UNG Mix
2. 2 × SensiDirect Premix Buffer (dUTP)
Ọnọdụ nchekwa
All components kwesịrị idobe na -20 ℃ maka nchekwa ogologo oge yana 4 ℃ ruo ọnwa 3.Biko gwakọta nke ọma mgbe ịchasịrị na centrifuge tupu i jiri ya.Zere na-agbaze oyi mgbe niile.
Protocol ịgba ígwè
| Nzọụkwụ | Okpomọkụ | Oge | okirikiri |
| mgbaze | 50 ℃ | Nkeji 2 | 1 |
| Polymerase ịgbalite | 95 ℃ | 1-5 nkeji | 1 |
| Denature | 95 ℃ | 10-20s | 40-50 |
| Mwepu / Mgbatị | 56-64 ℃ | Afọ 20-60 |
Ntuziaka pipet
| Reagent | Olu kwa mmeghachi omume | Olu kwa mmeghachi omume | Ntụkwasị obi ikpeazụ |
| 2 × SensiDirect Premix Buffer (dUTP) | 12.5µL | 25µL | 1× |
| 50× SensiDirect Enzyme/UNG Mix | 0.5µL | 1µL | 1× |
| Ngwakọta 25 × Primer-Probe Mix1, 2 | 1µL | 2µL | 1× |
| Ihe atụ3, 4 | - | - | - |
| ddH2O | - | - | - |
| Mkpokọta olu | 25 μl | 50 μl | - |
1. Nkwenye ikpeazụ nke primer na-abụkarị 0.2μM.Maka nsonaazụ kacha mma, enwere ike ịkwalite ntinye nke primer n'ime oke nke 0.2-1μM.
2. N'ozuzu, nyocha nyocha nwere ike kachasị mma n'ime oke nke 0.1-0.3μM.Nleba kachasị mma nke nyocha ahụ metụtara ngwa ngwa PCR n'ezie, ụdị nyocha, na ụdị ihe akara akara fluorescent.Biko rụtụ aka na ntuziaka akụrụngwa ma ọ bụ ihe achọrọ maka nyocha ọ bụla nke fluorescent.
3. Ụdị ihe atụ dị iche iche nwere ụdị dị iche iche na ọdịnaya nke inhibitor na nọmba nke mkpụrụ ndụ ihe mgbaru ọsọ.Ekwesịrị ịtụle olu nlele ahụ site na ọnọdụ n'ezie.Mee dilution nke ihe nlele site na ịgbakwunye mmiri na-enweghị nuclease ma ọ bụ TE Buffer, ọ bụrụ na ọ dị mkpa.
4. akwadoro olu nke ụdị dị iche iche:
| Ihe atụ | Olu maka otu 50 μL mmeghachi omume | Kachasị oke |
| Ọbara zuru oke nke anticoagulated | 2.5 μl | 5% |
| Plasma | 15 μl | 30% |
| Ọbara | 10 μl | 20% |
| Akpịrị swab | 10 μl | 20% |
| Saliva | 10 μl | 20% |
Njikwa ogo
1. Nchọpụta ọrụ: uche, nkọwapụta na nkwughachi nke qPCR.
2. Ọ dịghị ọrụ nuclease exogenous: ọ dịghị exogenous endonuclease na exonuclease mmetọ.
Ihe ndetu nke ngwaahịa
1. Ngwaahịa a na-eji ụdị ọhụrụ nke DNA polymerase na-ekpo ọkụ na-ekpo ọkụ, nke nwere ike ime ka ọ rụọ ọrụ na nkeji 1-5. Ebe ọ bụ na emeziwanye ihe nkwụnye mmeghachi omume ya, ọ dị mma maka PCR abụọ ma ọ bụ ọtụtụ fluorescence quantitative PCR site na iji usoro nyocha.
2. Ọ bụrụ na Rn uru nke PCR amplification dị oke ala ma ọ bụ a na-egbochi nkwalite ahụ, na-ebelata ego nlele, ịba ụba mmeghachi omume ma ọ bụ dilution gara aga nke ihe nlele ahụ nwere ike ime ka nsonaazụ ya dịkwuo mma.
3. Nchịkọta nke ọbara, mmiri mmiri, mmamịrị, akpịrị akpịrị, wdg kwesịrị ịgbaso ụkpụrụ ụlọ ọgwụ chọrọ, enwere ike iji ihe nlele ọhụrụ mee ihe iji gbochie mmebi nucleic acid.
4. Ebe ọ bụ na amplicon dị iche iche nwere dị iche iche itinye n'ọrụ arụmọrụ ka dUTP na uche na UNG, na reagents kwesịrị kachasị ma ọ bụrụ na nchọpụta uche na-ebelata mgbe eji usoro UNG.Biko kpọtụrụ anyị maka nkwado teknụzụ ma ọ bụrụ na ọ dị mkpa.
5. Iji zere mmụba nke ngwaahịa PCR na-ebufe n'etiti mmeghachi omume otu nzọụkwụ, mpaghara nnwale raara onwe ya nye na pipette chọrọ maka mmụba.Jiri uwe aka ma na-agbanwe ugboro ugboro ma emeghela tube mmeghachi omume mgbe nkwalite PCR gasịrị.
