Difference between revisions of "Part:BBa K2889003:Design"

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We synthesized miR-21-sponge-4s from BBa_K2514000.
 
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
 
https://static.igem.org/mediawiki/parts/e/e6/Amplification_of_miR-21-sponge-4s_fragment.jpeg
  
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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.
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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  
 
https://static.igem.org/mediawiki/parts/5/5b/Digested_the_PSB1C3_vectors_with_EcoRI_and_PstI.jpeg  
  
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We digested the PSB1C3 vectors with restriction enzymes EcoRI and PstI
+
 
 +
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
 
https://static.igem.org/mediawiki/parts/1/1d/MiR-21-sponge-4s_sequencing_map.jpeg
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miR-21-sponge-4s fragments were ligated to PSB1C3 vector. Then we selected the positive clones by PCR and sequencing
 
  
 
===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
 
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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 66
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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)


Amplification_of_miR-21-sponge-4s_fragment.jpeg


2.We digested the PSB1C3 vectors with restriction enzymes EcoRI and PstI. (Fig. 2)


Digested_the_PSB1C3_vectors_with_EcoRI_and_PstI.jpeg


3.miR-21-sponge-4s fragments were ligated to PSB1C3 vector. Then we selected the positive clones by PCR and sequencing.(Fig. 3)



MiR-21-sponge-4s_sequencing_map.jpeg

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