Difference between revisions of "Part:BBa K2889003:Design"
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===Design Notes=== | ===Design Notes=== | ||
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| + | In our previous project, we constructed two miR-21 sponge plasmid, pEGFP-miR-21-sponge-2s and pEGFP-miR-21-sponge-6s, as the monitor to detect the expression of miR-21 in cells.The slope of the standard curve of miR-21-sponge-2s is better than miR-21-sponge-6s, suggesting the more sensitive of miR-21-sponge-2s as a monitor. This year, we wanted to improve the sensitive of miRNA sponge. We designed miR-21 sponges contains four miR-21 binding sites with 3-nt spacers for bulged sites based on the sequence of hsa-miR-21 according to the previous study.We constructed pEGFP-miR-21-sponge-4s and pSB1C3-miR-21-sponge-4s this year. Our data suggested miR-21-sponge-4s is more sensitive than miR-21-sponge-2s and miR-21-sponge-6s. | ||
===Source=== | ===Source=== | ||
| − | + | We synthesized miR-21-sponge-4s from BBa_K2514000. | |
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| + | 1.First we amplified miR-21-sponge-4s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes EcoR I and Pst I.(Fig. 1) | ||
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| + | https://static.igem.org/mediawiki/parts/e/e6/Amplification_of_miR-21-sponge-4s_fragment.jpeg | ||
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| + | 2.We digested the PSB1C3 vectors with restriction enzymes EcoRI and PstI. (Fig. 2) | ||
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| + | https://static.igem.org/mediawiki/parts/5/5b/Digested_the_PSB1C3_vectors_with_EcoRI_and_PstI.jpeg | ||
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| + | 3.miR-21-sponge-4s fragments were ligated to PSB1C3 vector. Then we selected the positive clones by PCR and sequencing.(Fig. 3) | ||
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| + | https://static.igem.org/mediawiki/parts/1/1d/MiR-21-sponge-4s_sequencing_map.jpeg | ||
===References=== | ===References=== | ||
| + | Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods. 2007 Sep;4(9):721-6 | ||
Latest revision as of 12:16, 11 October 2018
pSB1C3-miR-21-sponge-4s
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 66
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
In our previous project, we constructed two miR-21 sponge plasmid, pEGFP-miR-21-sponge-2s and pEGFP-miR-21-sponge-6s, as the monitor to detect the expression of miR-21 in cells.The slope of the standard curve of miR-21-sponge-2s is better than miR-21-sponge-6s, suggesting the more sensitive of miR-21-sponge-2s as a monitor. This year, we wanted to improve the sensitive of miRNA sponge. We designed miR-21 sponges contains four miR-21 binding sites with 3-nt spacers for bulged sites based on the sequence of hsa-miR-21 according to the previous study.We constructed pEGFP-miR-21-sponge-4s and pSB1C3-miR-21-sponge-4s this year. Our data suggested miR-21-sponge-4s is more sensitive than miR-21-sponge-2s and miR-21-sponge-6s.
Source
We synthesized miR-21-sponge-4s from BBa_K2514000.
1.First we amplified miR-21-sponge-4s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes EcoR I and Pst I.(Fig. 1)
2.We digested the PSB1C3 vectors with restriction enzymes EcoRI and PstI. (Fig. 2)
3.miR-21-sponge-4s fragments were ligated to PSB1C3 vector. Then we selected the positive clones by PCR and sequencing.(Fig. 3)
References
Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods. 2007 Sep;4(9):721-6