Otu nzọụkwụ RT-qPCR SYBR Green Premix
Mpempe nwamba: HCB5140A
Otu nzọụkwụ RT-qPCR Syber Green Premix bụ maka ọnụọgụ fluorescence dabere na SYBR Green I dye.N'iji mkpụrụ ndụ ihe nketa kpọmkwem, ntụgharị ntụgharị na mmeghachi omume qPCR na-agwụcha n'otu tube, na-ewepụ mkpa maka imeghe okpu ugboro ugboro na ọrụ ọkpọkọ, na-eme ka arụmọrụ nyocha dịkwuo mma na ibelata ihe ize ndụ nke mmetọ.Maka nlele RNA, ngwa ahụ na-eji Reverse Transcriptase na-eguzogide okpomọkụ maka njikọ cDNA nke ọma yana HotStart Taq DNA Polymerase maka mmụba ọnụọgụ.N'okpuru sistemụ nchekwa kachasị mma, ọgụgụ isi nke ngwa ahụ nwere ike ịdị elu dị ka 0.1 pg maka ebumnuche egosipụtara nke ukwuu yana ihe ruru 1 pg maka ebumnuche egosipụtara nke ọma.Ngwa ahụ dabara maka mmụba na ọnụọgụ nke ihe nlele DNA.Ọ na-enyere aka ịchọpụta na ọnụọgụ nucleic acid sitere na ihe atụ osisi na anụmanụ dị iche iche, sel na microorganisms.
Ngwa
No | Aha | Olu | Olu |
1 | Ihe nchekwa dị elu | 250 μl | 2 × 1.25 ml |
2 | Ngwakọta Enzyme dị elu | 20 μl | 200 μl |
3 | RNase Free H2O | 250 μl | 2 × 1.25 ml |
Ọnọdụ Nchekwa
Ekwesịrị ịchekwa ngwaahịa a na -25 ~ -15 ℃ pụọ na ìhè maka otu afọ.
Ntuziaka
1.Reaction nhazi usorod
Ngwa | Olu (μL) | Olu (μL) | Ntinye uche ikpeazụ |
Ihe nchekwa dị elu | 12.5 | 25 | 1× |
Ngwakọta Enzyme dị elu | 1 | 2 | - |
Ndị na-aga n'ihu (10 μmol/L)a | 0.5 | 1 | 0.2 μmol / L |
Agbaghachi azụ (10 μmol/L)a | 0.5 | 1 | 0.2 μmol / L |
RNA Tamplateb | X | X | - |
RNase Free H2Oc | ruo 25 | ruo 50 | - |
ndetu:
1) a.TNtinye uche nke ikpeazụ bụ 0.2 μmol/L, nke nwekwara ike gbanwee n'etiti 0.1 na 1μmol/L dị ka o kwesịrị.
2) b.The reagent nwere mmetụta nke ukwuu, yana ngụkọta RNA na oke nke 1pg-1μg, na nnwale nke ihe nlele mmadụ gosipụtara ntinye kacha mma nke 1 pg-100 ng, na-achịkwa maka uru Ct n'ozuzu ya na oke 15-30 dị ka o kwesịrị.
3) c.A na-atụ aro ka iji 20μL ma ọ bụ 50μL iji hụ na ịdị irè na nrụpụta nke nkwalite mkpụrụ ndụ ihe mgbaru ọsọ.
4) d.Biko kwadebe n'oche oche dị ọcha ma jiri ndụmọdụ na-enweghị nuclease na tubes mmeghachi omume;A na-atụ aro ndụmọdụ nwere katrij nzacha.Zere mmetọ obe na mmetọ ikuku.
2.Mmemme mmeghachi omume
Nzọụkwụ okirikiri | Okpomọkụ | Oge | okirikiri |
Ntugharị azụ azụ | 50 ℃a | 6mins | 1 |
denaturation nke mbụ | 95 ℃ | 5mins | 1 |
Mmeghachi omume nkwalite | 95 ℃ | 15 nkeji | 40 |
60 ℃b | 30 sk | ||
Agba agbaze okirikiri | Ngwa akụrụngwa | 1 |
ndetu:
1) a.Enwere ike ịhọrọ okpomọkụ ntụgharị ntụgharị n'etiti 50-55 Celsius dịka mkpa nnwale siri dị.Maka nlele DNA, enwere ike ịhapụ usoro ntụgharị ntụgharị.
2) b.N'ọnọdụ ndị pụrụ iche enwere ike imezi okpomọkụ mgbakasị ahụ / mgbatị ahụ dịka uru Tm primer si dị, a na-atụ aro 60 Celsius.
Ihe ndetu
1. Ngwaahịa a bụ maka nyocha naanị.
2. Biko rụọ ọrụ na uwe lab na uwe mwụfu, maka nchekwa gị.