M-MLV Neoscript Reverse Transcriptase
Ko te Neoscript Reverse Transcriptase he transcriptase whakamuri i whiwhi ma te tirotiro i te ira ira M-MLV o Moloney murine leukemia huaketo takenga mai me te whakapuaki i roto i te E.coli.Ka whakakorehia e te enzyme te mahi RNase H, he teitei ake te whakamaaramatanga o te pāmahana, he pai hoki mo te whakamaoritanga whakamuri-nui.Na reira, he awhina mo te whakakore i nga paanga kino o te hanganga taumata-tiketike RNA me nga mea kore-motuhake i runga i te cDNA synthesis, me te kaha ake o te pumau me te kaha o te whakawhitinga whakawhiti.He teitei ake te pumau o te enzyme me te kaha ki te whakahiato whakamuri.
Waehanga
1.200 U/μL Neoscript Reverse Transcriptase
2.5 × Pūreirei-Tuatahi (he kōwhiri)
* 5 × Karekau he dNTP i roto i te Whero Tuatahi, taapirihia nga dNTP ina whakarite ana i te punaha tauhohenga.
Taupānga Manakohia
1.Kotahi-taahiraa qRT-PCR.
2.Te kitenga huaketo RNA.
Tikanga Rokiroki
-20°C mo te rokiroki mo te wa roa, me uru pai i mua i te whakamahi, karohia te whakamao-kore-roa.
Whakamaramatanga Waeine
Kotahi te waeine e whakauru ana i te 1 nmol o te dTTP i roto i te 10 meneti i te 37°C ma te whakamahi i te poly(A)•oligo(dT)25hei tauira/primer.
Mana Kounga
1.SDS-PAGE electrophoretic purenga nui ake i te 98%.
2.Te tairongo whakakaha, te mana o te puranga-ki-te-puranga, te pumau.
3.Karekau he mahi karihi o waho, karekau he endonuclease o waho, he poke exonuclease ranei
Tauhohenga Tauhohenga mo te Otinga Hohenga Hohenga Tuatahi
1.Te whakarite i te ranunga tauhohenga
| Waehanga | Rōrahi |
| Oligo(dT)12-18 Primer Tuhinga o mua raneia Ranei Gene Specific Primersb | 50 pmol |
| 50 pmol (20-100 pmol) | |
| 2 pmol | |
| 10 mM dNTP | 1 μL |
| RNA tauira | Tapeke RNA≤ 5μg;mRNA≤ 1 μg |
| RNase-kore dH2O | Ki te 10 μL |
Tuhipoka:a/b: Tīpakohia ngā momo tuatahi kia rite ki o hiahia whakamatautau.
2.Whakawerahia i te 65°C mo te 5 meneti ka whakamatao tere ki te hukapapa mo te 2 meneti.
3.Tāpirihia nga waahanga e whai ake nei ki te punaha i runga ake nei ki te 20µL katoa ka uru marie:
| Waehanga | Rōrahi (μL) |
| 5 × Paerewa-Tuatahi | 4 |
| Neoscript Reverse Transcriptase (200 U/μL) | 1 |
| Te aukati RNase (40 U/μL) | 1 |
| RNase-kore dH2O | Ki te 20 μL |
4.Whakamahia te tauhohenga kia rite ki nga tikanga e whai ake nei:
(1) Mena ka whakamahia te Random Primer, me kawe te tauhohenga ki te 25 ℃ mo te 10mins, ka 50 ℃ mo te 30 ~ 60mins;
(2) Mēnā ka whakamahia te Oligo dT me ngā pūmua motuhake, me kawe te tauhohenga ki te 50 ℃ mō te 30~60mins.
5.Whakawerahia i te 95 ℃ mo te 5 meneti hei whakakore i te Neoscript Reverse Transcriptase me te whakamutu i te tauhohenga.
6.Ka taea te whakamahi tika i nga hua tuhi whakamuri i roto i te tauhohenga PCR me te tauhohenga PCR ine ine, ka rongoa ranei ki te -20 ℃ mo te wa roa.
PCR Rmahi:
1.Te whakarite i te ranunga tauhohenga
| Waehanga | Te aro |
| 10 × PCR Buffer (dNTP kore utu, Mg²+ kore utu) | 1× |
| dNTP (10mM ia dNTP) | 200 μM |
| 25 mM MgCl2 | 1-4 mM |
| Taq DNA Polymerase (5U/μL) | 2-2.5 U |
| Tuatahi 1 (10 μM) | 0.2-1 μM |
| Tuatahi 2 (10 μM) | 0.2-1 μM |
| tauiraa | ≤10% Rongoa Tauhohenga Tiini Tuatahi (2 μL) |
| ddH2O | Ki te 50 μL |
Tuhipoka:a: Mena he nui rawa te otinga tauhohenga mekameka tuatahi ka taapirihia, ka taea te aukati i te tauhohenga PCR.
2.Te Tikanga Tauhohenga PCR
| Hipanga | Te pāmahana | Te wa | Nga huringa |
| Tuhinga o mua | 95℃ | 2-5 min | 1 |
| Te whakahekenga | 95℃ | 10-20 hēkona | 30-40 |
| Te whakaene | 50-60 ℃ | 10-30 hēkona | |
| Toronga | 72℃ | 10-60 hēkona |
Notes
1.He pai mo te whakamaarama whakamaarama whakamaaramatanga whakamuri i roto i te awhe o te 42 ℃~55 ℃.
2.He pai ake te pumau, he pai mo te whakamaarama whakamuri whakamuri whakamaarama teitei.I tua atu, he pai mo te haere pai i roto i nga rohe hanganga matatini o te RNA.Ano, kohe pai mo te kotahi-step multiplex fluorescence ine RT-PCR rapunga.
3.He pai te hototahitanga ki nga momo whaariki whakakaha PCR me te pai mo nga tauhohenga RT-PCR tairongo teitei.
4.E tika ana mo te tairongo teitei kotahi-taahiraa fluorescence ine ine RT-PCR tauhohenga, tōtika te whakapai ake i te auau rapunga o te kukū iti o tauira.
5.He pai mo te hanga whare pukapuka cDNA.
