Superstart qPCR Premix plus-UNG
Pusi Numera: HCB5071E
Superstart qPCR Premix plus-UNG ose reagent faʻapitoa ua fuafuaina mo le taimi moni PCR faʻataʻitaʻiga lelei ma faʻatusatusaga e faʻaaoga ai suʻesuʻega faʻavae suʻesuʻe, na atiaʻe faapitoa mo faiga lyophilization.O loʻo i ai se enzyme Hotstart Taq plus (DG), o loʻo faʻamaufaʻailogaina le gaioiga o le enzyme Taq i le vevela o le potu, faʻamalosia lelei le faʻalavelaveina o le faʻalauteleina e le faʻapitoa e mafua mai i le faʻapipiʻiina e le faʻapitoa poʻo le faʻavaeina o le dimer i lalo o tulaga maualalo o le vevela, ma faʻaleleia atili ai. le fa'apitoa o le fa'atupu fa'ateleina.O lenei reagent e faʻaogaina le faʻaogaina o le qPCR faʻapitoa ma le UNG / dUTP anti-contamination system e ausia ai le vave vevela-amata, faʻaleleia atili le lelei ma le maaleale o tali qPCR.E mafai ona maua curves tulaga lelei i le tele o vaega quantitation ma saʻo faatino quantitation, lelei puipuia amplification lelei sese e mafua mai oloa PCR totoe po o aerosol contamination.O lenei reagent e fetaui lelei ma le tele o meafaifaʻaili PCR faʻataʻitaʻiga mai tagata gaosi oloa e pei ole Applied Biosystems, Eppendorf, Bio-Rad ma Roche ma isi, ma faʻaalia lelei le mautu ile lyophilized form.
Reagent fatuga
1. 5×HotstartPremix faaopoopo-UNG (Mg2+fua) (DG)
2. 250 mM MgCl2
3. 4×lyoprotectant (filifiliga)
Tulaga Teuteu
Taofi umi i le -20℃;e mafai ona teuina i le 4 ℃ mo le 3 masina.Faafefiloi lelei a o lei faaaogaina maaloese mai le fa'a'aisa fa'alua ma le liu.
Polokalama uila uila
| Taualumaga | Temp. | Taimi | Ta'amilosaga |
| ga'o | 50℃ | 2 minu | 1 |
| Fa'atosinaina o le Polymerase | 95℃ | 1~5 min | 1 |
| Denature | 95℃ | 10~20 s | 40-50 |
| Su'ega ma Fa'aopoopo | 56~64 ℃ | 20~60 s | 40-50 |
qPCR Liquid Reaction Syla'au Sauniuniga
|
Tulaga |
25µL Volume |
50µL Volume |
Fa'ai'uga Fa'amau |
| 5×HotstartPremix faaopoopo-UNG(Mg2+fua) (DG) | 5µL | 10µL | 1× |
| 250mM MgCl2 | 0.45µL | 0.9µL | 4.5 mM |
| 4×lyoprotectant1 | 6.25µL | 12.5µL | 1× |
| 25×Primer-Su'esu'e Fa'afefiloi2 | 1µL | 2µL | 1× |
| DNA mamanu3 | —— | —— | —— |
| ddH2O | I le 25µL | I le 50µL | —— |
1. O se faʻasalaga mulimuli o le 0.2μM mo faʻamuamua e masani ona maua ai iʻuga lelei;pe a le lelei le faatinoga o tali, fetuutuuna'i le fa'atonuga muamua i totonu ole va ole 0.2-1μM pe a mana'omia.Ole fa'atonuga ole su'esu'ega e masani lava ona fa'asili ile va ole 0.1-0.3μM e ala ile fa'ata'ita'iga gradient e su'e ai tu'ufa'atasiga sili ona lelei.
2. O le numera kopi o kene fa'atatau o lo'o i totonu o ituaiga fa'ata'ita'iga e eseese;pe a mana'omia, e mafai ona faia le fa'afefiloi o le gradient e fuafua ai le aofa'i fa'aopoopo fa'atusa sili ona lelei.
3. O lenei faiga e mafai ona lyophilized;pe a faʻaaogaina e le au faʻatau lenei faiga e aunoa ma le faʻamago faʻamago manaʻoga, 4 × lyoprotectant e mafai ona faʻapipiʻi faʻaopoopo; afai o loʻo i ai ni mea faʻamago faʻamago e manaʻomia, i le taimi o le faʻaogaina o le vai e faʻamaonia ai le faʻatinoga o oloa, e tatau ona faʻaopoopoina le 4 × lyoprotectant e faʻamautinoa le ogatasi ma vaega o le lyophilized. ma aafiaga.
A fa'aoga le faigamo le fa'a'aisa-fa'agogo, saunia le faiga as o lo'o mulimuli mai:
| Tulaga | 25µL Reaction System |
| 5 ×HotstartPremix faaopoopo-UNG (Mg2+fua) (DG) | 5µL |
| 250mM MgCl2 | 0.45µL |
| 4×lyoprotectant | 6.25µL |
| 25×Primer-Su'esu'e Fa'afefiloi | 1µL |
| ddH2O | I le 18~20µL |
* Afai e mana'omia nisi faiga mo le fa'a'aisa fa'amago, fa'amolemole fa'afeso'ota'i ese.
Lyophilization Taualumagass
| Taualumaga | Temp. | Taimi | Tulaga | Omiga |
| Mu'a'aisa | 4℃ | 30 min | Taofi |
1 atm |
| -50℃ | 60 min | Fa'alili | ||
| -50℃ | 180 min | Taofi | ||
| Fa'agogo Tulaga Muamua | -30℃ | 60 min | Fa'avevela |
Vacuum Sili |
| -30℃ | 70 min | Taofi | ||
| Faamago Lua | 25℃ | 60 min | Fa'avevela |
Vacuum Sili |
| 25℃ | 300 min | Taofi |
2. O le faagasologa lyophilization o loʻo i luga e mo naʻo le faʻamatalaga.O ituaiga oloa eseese ma eseese fa'a'aisa-mamago e eseese ta'iala, o lea e mafai ai ona faia fetuunaiga e tusa ai ma le mea moni.tulaga i le taimi o le faaaogaina.
3. Eseese lyophilization faiga e mafai ona talafeagai mo le tele o faaputuga lyophilized eseeseoloa, o lea e tatau ona lava le faʻamaoniga o suʻega pe a faʻaaogaina mo le gaosiga tele.
Fa'atonuga mo le fa'aogaina o le lyophilized pauta
1. Centrifuge puupuu le pauta lyophilized;
2. Fa'aopoopo le fa'ata'ita'iga o le nucleic acid i le pa'u lyophilized ma fa'aopoopo le vai i le 25µL;
3. Faafefiloi lelei e ala ile centrifugation ma tamo'e ile masini.
Pulea lelei:
1. Suʻega faʻatinoga: faʻalogona, faʻapitoa, toe faʻaleleia o le qPCR.
2. Leai se gaioiga fa'atosina mai fafo, leai se fa'aleagaina o le endo/exonuclease.
Fa'amatalaga Fa'atekinisi:
1. Superstart qPCR Premix plus-UNG e faʻaaogaina se enzyme vevela-amata e mafai ai ona vave vevela-amata i totonu ole 1 ~ 5 minute;e ala i le fa'apolopolo fa'apitoa e fetaui lelei mo le tele o fa'alili ole PCR fa'atatau.
2. E i ai tulaga faapitoa maualuga lea e matua faaleleia ai le lagona o le fluorescence quantitative PCR tapulaa suʻesuʻega, faia amplification curves normalization, fluorescence tau maua le faaleleia manino i templates concentration maualalo, talafeagai e pei o le maualuga maaleale fluorescence quantitative PCR detection reagents.
3. Mo primers e maualalo le vevela vevela poʻo umi atu nai lo 200bp fasi vaega, 3-laasaga auala e fautuaina.
4. O le faʻaogaina lelei o le dUTP ma le faʻaogaina o le UNG enzyme e eseese mo kenera eseese, ma afai o le faʻaogaina o le UNG system e oʻo atu ai i le faʻaitiitia o le faʻaogaina o le faʻaogaina, e tatau ona fetuunaʻi ma faʻaleleia le faiga faʻaalia.Afai e mana'omia le lagolago fa'apitoa fa'amolemole fa'afeso'ota'i la matou kamupani.
5. Fa'aaogā vaega fa'apitoa ma paipa a'o le'i fa'ateleina ma pe a uma fo'i, fai totigilima a'o fa'agaioiga ma sui soo;'aua ne'i tatalaina le paipa fa'afefe pe a mae'a le PCR e fa'aitiitia ai le fa'aleagaina o fa'ata'ita'iga e oloa PCR.
