Team kansai protocols: Difference between revisions
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== <font face="Helvetica" color="#298cda">Materials</font> == | |||
<p style="line-height: 200%"> <font face="Helvetica"> | |||
Staple DNA strands were purchased from Sigma Genosys (Hokkaido, Japan) and used without further purification.</font> </p> | |||
== <font color="#298cda"> | == <font face="Helvetica" color="#298cda">Self-assembly of DNA origami (Stage, convex DNA origami)</font> == | ||
<p style="line-height: 200%"> <font face="Helvetica"> | |||
The formation of DNA origami was performed with M13mp18 ssDNA (4 nM, Takara, Japan), staple strands (20 nM for each strand) in a solution containing Tris (40 mM), acetic acid (20 mM), EDTA (10 mM), and magnesium acetate (12.5 mM, 1 X TAE/Mg buffer, 50 µL). This mixture was cooled from 90˚C to 25˚C at a rate of -1.0˚C/min to anneal the strands. | |||
</font> </p> | |||
== <font color="#298cda"> | == <font face="Helvetica" color="#298cda">Combine Stage with convex DNA origami</font> == | ||
<p style="line-height: 200%"> <font face="Helvetica"> | |||
Two DNA origami solution, Stage and convex DNA origami, were mixed into a microtube. The mixture was annealed from 40℃ to room temperature slowly. | |||
</font> </p> | |||
== AFM observation == | == <font face="Helvetica" color="#298cda">AFM observation</font> == | ||
<p style="line-height: 200%"> <font face="Helvetica"> | |||
AFM imaging of DNA origami was performed on a E-sweep system (SII, Japan). The mixture (1 µL) was deposited on freshly cleaved mica, additional 1X TAE/Mg buffer (200 µL) was added, and the imaging was performed in the fluid DFM scanning mode with a BL-AC40TS tip (Olympus, Japan). | |||
</font> </p> | |||
== <font face="Helvetica" color="#298cda">Agarose gel electrophoresis</font> == | |||
<p style="line-height: 200%"> <font face="Helvetica"> | |||
The assembled products were loaded into agarose gels (1.5% agarose in 1×TAE/Mg<sup>2+</sup> aqueous buffer) and subject to gel electrophoresis at 100 V for one hour. The gel was stained with GelStar. | |||
</font> </p> | |||
Latest revision as of 17:07, 30 October 2011
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Materials
Staple DNA strands were purchased from Sigma Genosys (Hokkaido, Japan) and used without further purification.
Self-assembly of DNA origami (Stage, convex DNA origami)
The formation of DNA origami was performed with M13mp18 ssDNA (4 nM, Takara, Japan), staple strands (20 nM for each strand) in a solution containing Tris (40 mM), acetic acid (20 mM), EDTA (10 mM), and magnesium acetate (12.5 mM, 1 X TAE/Mg buffer, 50 µL). This mixture was cooled from 90˚C to 25˚C at a rate of -1.0˚C/min to anneal the strands.
Combine Stage with convex DNA origami
Two DNA origami solution, Stage and convex DNA origami, were mixed into a microtube. The mixture was annealed from 40℃ to room temperature slowly.
AFM observation
AFM imaging of DNA origami was performed on a E-sweep system (SII, Japan). The mixture (1 µL) was deposited on freshly cleaved mica, additional 1X TAE/Mg buffer (200 µL) was added, and the imaging was performed in the fluid DFM scanning mode with a BL-AC40TS tip (Olympus, Japan).
Agarose gel electrophoresis
The assembled products were loaded into agarose gels (1.5% agarose in 1×TAE/Mg2+ aqueous buffer) and subject to gel electrophoresis at 100 V for one hour. The gel was stained with GelStar.
