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[求助]
RNA与镁离子
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求助下面这段描述中的镁离子是有什么作用的？另外镁离子与RNA的稳定与构象有关吗？原理是什么呢？ A T7 primer and gene-specific primers were used to PCR amplify pri-miR- 1792 sequences from plasmid DNA template. PCR products were gelpurified and used as templates for in vitro transcription using the Riboprobe (Promega) system together with 32P-CTP for radioactive labeling. Microprocessor purified from Flag-DROSHA-293 cells was used for in vitro Microprocessor assays (Gregory et al., 2004). Cold RNA was produced using the same strategy without 32P-CTP addition. For RNA annealing, 10 mM Sample Text was added to 200 pmol cold RNA and incubated at 95C for 5 min and then slowly cooled to reverse transcription (RT). Annealed RNA was subjected to 5% native polyacrylamide gel for ethidium bromide staining and used for Microprocessor assay followed by small RNA northern blot analysis. His- CPSF3 complex was purified from E. coli as described previously for other proteins (Chang et al., 2013). Assays conditions were as for Microprocessor. |
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In Vitro Transcription, Microprocessor, and CPSF3 Cleavage Assays A T7 primer and gene-specific primers were used to PCR amplify pri-miR- 1792 sequences from plasmid DNA template. PCR products were gelpurified and used as templates for in vitro transcription using the Riboprobe (Promega) system together with 32P-CTP for radioactive labeling. Microprocessor purified from Flag-DROSHA-293 cells was used for in vitro Microprocessor assays (Gregory et al., 2004). Cold RNA was produced using the same strategy without 32P-CTP addition. For RNA annealing, 10 mM MgCl2 was added to 200 pmol cold RNA and incubated at 95C for 5 min and then slowly cooled to reverse transcription (RT). Annealed RNA was subjected to 5% native polyacrylamide gel for ethidium bromide staining and used for Microprocessor assay followed by small RNA northern blot analysis. His- CPSF3 complex was purified from E. coli as described previously for other proteins (Chang et al., 2013). Assays conditions were as for Microprocessor. |
2楼2015-09-23 18:37:14
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