M-MLV Reverse Transcriptase
Loaʻa ka RevScript Reverse transcriptase e ka ʻenehana ʻenehana genetic.Loaʻa iā ia ka mana synthesis cDNA kiʻekiʻe, ke kūpaʻa wela a me ka palena wela o ka hopena (a hiki i 60°C).ʻO ka huahana cDNA synthesized a hiki i 10 kb.Hoʻonui ia i ka pilina o nā mamana a kūpono no ka hoʻohuli ʻana i nā mamana RNA me ka paʻakikī paʻakikī a i ʻole nā genes kope haʻahaʻa.
Nā ʻāpana
| ʻāpana | HC2003B-01 (10,000U) | HC2003B-02 (5*10,000U) | HC2003B-03 (200,000U) |
| RevScript Reverse Transcriptase (200U/μL) | 50 μL | 5×50 μL | 1 mL |
| 5 × RevScript Buffer | 250 μL | 1.25 mL | 5 mL |
Kūlana Waihona
Pono e mālama ʻia kēia huahana ma -25°C~-15°C no 2 mau makahiki.
Wehewehe Unite
Hoʻokahi pūʻulu hoʻohui i 1 nmol o dTTP i loko o nā mea hiki ʻole ke hoʻoheheʻe ʻia i loko o 10 mau minuke ma 37°C me ka hoʻohana ʻana iā Oligo(dT) ma ke ʻano he kumu mua.
Hoʻonohonoho ʻana
1.Denaturation o ka RNA template (He mea koho keia pae, denaturation o RNA template kokua i ka wehe ana i ka lua o ka hale, e hoonui i ka hua o ka mua strand cDNA.)
| Nā ʻāpana | Volume (μL) |
| RNase manuahi ddH2O | I ka 13 |
| Oligo(dT)18 (50 μmol/L) a i ʻole ke kumu mua (50 μmol/L) A i ʻole Gene Specific Primers (2 μmol/L) | 1 |
| a i ʻole 1 | |
| a i ʻole 1 | |
| RNA template | Xa |
Nā memo:
1) a: Ka huina RNA: 1-5 ug a i ʻole mRNA: 1-500 ng
2) Hoʻomoʻa ʻia ma 65 ° C no 5 mau minuke, a laila e hoʻololi koke i ka hau e hoʻomaha no 2 mau minuke.ʻO ka centrifugation pōkole e hōʻiliʻili i ka wai hopena, e hoʻohui i ka hopena hoʻololi hoʻololi e like me ka mea i hōʻike ʻia ma ka papa ma lalo.E hoʻopili mālie i ka pipette.
1. Ka hoʻomākaukau ʻana i ka hui ʻana (20 μL volume)
| Nā ʻāpana | Volume (μL) |
| Huina o ka hana mua | 13 |
| 5×Buffer | 4 |
| Huihui dNTP (10nmol/L) | 1 |
| Transcriptase Reverse (200 U/μL) | 1 |
| RNase inhibitor (40 U/μL) | 1 |
1.Hana i ka hopena ma lalo o kēia mau kūlana:
| Mahana (°C) | Manawa |
| 25 °Ca | 5 min |
| 42 °Cb | 15-30min |
| 85 °Cc | 5 min |
Nā memo:
1) a.Pono ka hoʻoulu ʻana ma 25 ° C no 5 mau minuke no ka hoʻohana ʻana i nā hexamers maʻamau.E ʻoluʻolu e lele i kēia kaʻina i ka wā e hoʻohana ai iā Oligo (dT)18a i ʻole Gene Specific Primer.
2) b.He 42°C ka mahana o ka palapala hoohuli hou, No na la'ana me na hale pa'akikī a i 'ole maona GC ki'eki'e, ua paipai 'ia e ho'oki'eki'e i ke ana wela i 50-55°C.
3) c.Hoʻomaʻamaʻa ma 85 ° C no 5 mau minuke e hoʻopau i ka transcriptase hoʻohuli.
4) Hiki ke hoʻohana pololei ʻia ka huahana i ka PCR a i ʻole qPCR reactions, a i ʻole mālama ʻia ma -20 ° C no ka mālama pōkole.Manaʻo ʻia e hoʻokaʻawale i nā huahana a mālama i -80 ° C no ka mālama lōʻihi.Hōʻalo i ka hoʻoheheʻe pinepine ʻana.
5) Ua kūpono ka huahana no hoʻokahi-step RT-qPCR, ua ʻōlelo ʻia e hoʻohui i ka 10-20 U reverse transcriptase no kēlā me kēia 25μL reaction system, a i ʻole e hoʻonui i ka nui o ka transcriptase reverse e like me ke kūlana maoli.
Nā memo
1.E ʻoluʻolu e hoʻomaʻemaʻe i ka wahi hoʻokolohua;Pono e hoʻohana ʻia nā mīkina lima maʻemaʻe a me nā masks i ka wā o ka hana.Pono nā mea hoʻohana a pau i hoʻohana ʻia i ka hoʻokolohua ʻo RNase manuahi e pale ai i ka haumia RNase.
2. Pono e hana ʻia nā kaʻina hana a pau ma ka hau e pale ai i ka hōʻino ʻana o RNA.
3. ʻO nā hōʻailona RNA kiʻekiʻe kiʻekiʻe e ʻōlelo ʻia e hōʻoia i ka pono kiʻekiʻe o ka transcription hoʻohuli.
4. No ka noiʻi wale nō kēia huahana.
5. E ʻoluʻolu e hana me nā ʻaʻahu lab a me nā mīkina lima hoʻopau, no kou palekana.
