Team kansai result: Difference between revisions
No edit summary |
|||
| (29 intermediate revisions by the same user not shown) | |||
| Line 2: | Line 2: | ||
== <font face="Helvetica" color="#298cda">Stage</font> == | == <font face="Helvetica" color="#298cda">Stage</font> == | ||
[[Image:Stage AFM.png|thumb| | [[Image:Stage AFM.png|thumb|200px|<font face="Helvetica">Fig. 5. AFM images of (a) Stage, (b) Convex DNA origami and (c) Stage + Convex DNA origami. ([[Media:high resolution data.jpg|High resolution data of Stage + Convex DNA origami.]])</font>]] | ||
<p style="line-height: 200%"> <font face="Helvetica"> | <p style="line-height: 200%"> <font face="Helvetica"> | ||
First, We constructed the stage and the result is shown in Fig. | First, We constructed the stage and the result is shown in Fig. 5 (a). | ||
As you can see, the stage was successfully constructed. | As you can see, the stage was successfully constructed. | ||
However, most stages make complexes by blunt-end π–π stacking interactions. | |||
To suppress making complexes, thymine spacers were inserted in staple DNAs which is located at side of the stage. | To suppress making complexes, thymine spacers were inserted in staple DNAs which is located at side of the stage. | ||
</font> </p> | </font> </p> | ||
| Line 12: | Line 12: | ||
== <font face="Helvetica" color="#298cda">Convex DNA origami</font> == | == <font face="Helvetica" color="#298cda">Convex DNA origami</font> == | ||
<p style="line-height: 200%"> <font face="Helvetica"> | <p style="line-height: 200%"> <font face="Helvetica"> | ||
Next, we tried to construct the convex DNA origami and the result is shown in Fig. | Next, we tried to construct the convex DNA origami and the result is shown in Fig. 5 (b). | ||
Convex DNA origami was successfully constructed. | Convex DNA origami was successfully constructed. | ||
</font> </p> | </font> </p> | ||
| Line 19: | Line 19: | ||
<p style="line-height: 200%"> <font face="Helvetica"> | <p style="line-height: 200%"> <font face="Helvetica"> | ||
It was confirmed that Stage and Convex DNA origami were constructed. | It was confirmed that Stage and Convex DNA origami were constructed. | ||
Next, we tried to combine them. The result is shown in Fig. | Next, we tried to combine them. The result is shown in Fig. 5 (c) and confirmed that Stage was combined with Convex DNA origami which width was observed ~17 nm. | ||
At first time, the yield was ~20% and a reason of the low yield is that the mixture was only incubated at room temperature without slowly annealing. | |||
After this, Stage + Convex DNA origami was constructed with slowly annealing from 40℃ to room temperature and the yield rose up to ~60%. | |||
</font></p> | </font></p> | ||
== <font face="Helvetica" color="#298cda"> | == <font face="Helvetica" color="#298cda">Agarose gel electrophoresis</font> == | ||
[[Image:result of electrophoresis.png|thumb|200px|<font face="Helvetica">Fig. 6. The result of electrophoresis. Lane 1, M13mp18 ssDNA. Lane 2, Stage. Lane 3, Convex DNA origami. Lane 4, Stage + Convex DNA origami. ([[Media:raw data.jpg|Raw data]])</font>]] | |||
<p style="line-height: 200%"> <font face="Helvetica"> | |||
Formation of Stage + Convex DNA origami was confirmed by electrophoresis and the result is shown in Fig. 6. | |||
It was confirmed to construct Stage and Convex DNA origami because of the bands of Lane 2 (in red parentheses) and Lane 3 (in orange parentheses) have different electrophoretic mobility compared with Lane 1. | |||
A band having low mobility was observed in Lane 4 (in blue parentheses). | |||
It shows that Stage + Convex DNA origami was constructed. | |||
</font> </p> | |||
== <font face="Helvetica" color="#298cda">Formation of a molecular spider in a ditch</font> == | |||
[[Image:formation of molecular spider.png|thumb|200px|<font face="Helvetica">Fig. 7. AFM image of formation of molecular spider in a ditch</font>]] | |||
<p style="line-height: 200%"> <font face="Helvetica"> | |||
Then, we tried to form a molecular spider on the first scaffold. | |||
Fig. 7 is the result of it. There was few molecular spider in a ditch. We thought that streptavidin might be spoiled. | |||
</font> </p> | |||
== <font face="Helvetica" color="#298cda">Confirmation of forming a molecular spider</font> == | |||
<p style="line-height: 200%"> <font face="Helvetica"> | |||
We confirmed that streptavidin can bind with biotin by electrophoresis (Fig. 8). | |||
Lane 1 is the ratio of Streptavidin to biotinylated DNA equals the ratio of 1 to 1, Lane 2 is the ratio of 1 to 2, Lane 3 is the ratio of 1 to 3, Lane 4 is the ratio of 1 to 4. The higher the ratio of biotinylated DNA, the lower electrophoretic mobility the band became. This shows that streptavidin combines with biotinylated DNA regularly. | |||
</font> </p> | |||
[[Image:Confirmation of forming a molecular spider.png|thumb|200px|<font face="Helvetica">Fig. 8. PAGE analysis of streptavidin-biotinylated DNA conjugate.</font>]] | |||
Latest revision as of 06:25, 2 November 2011
<html><A href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Kansai"><IMG width="900px" src="http://openwetware.org/images/3/3b/Team_kansai_top.png"></A></html>
| Top | Team | Project | Results | Protocols | photos |
Stage
First, We constructed the stage and the result is shown in Fig. 5 (a). As you can see, the stage was successfully constructed. However, most stages make complexes by blunt-end π–π stacking interactions. To suppress making complexes, thymine spacers were inserted in staple DNAs which is located at side of the stage.
Convex DNA origami
Next, we tried to construct the convex DNA origami and the result is shown in Fig. 5 (b). Convex DNA origami was successfully constructed.
Stage + Convex DNA origami
It was confirmed that Stage and Convex DNA origami were constructed. Next, we tried to combine them. The result is shown in Fig. 5 (c) and confirmed that Stage was combined with Convex DNA origami which width was observed ~17 nm. At first time, the yield was ~20% and a reason of the low yield is that the mixture was only incubated at room temperature without slowly annealing. After this, Stage + Convex DNA origami was constructed with slowly annealing from 40℃ to room temperature and the yield rose up to ~60%.
Agarose gel electrophoresis
Formation of Stage + Convex DNA origami was confirmed by electrophoresis and the result is shown in Fig. 6. It was confirmed to construct Stage and Convex DNA origami because of the bands of Lane 2 (in red parentheses) and Lane 3 (in orange parentheses) have different electrophoretic mobility compared with Lane 1. A band having low mobility was observed in Lane 4 (in blue parentheses). It shows that Stage + Convex DNA origami was constructed.
Formation of a molecular spider in a ditch
Then, we tried to form a molecular spider on the first scaffold. Fig. 7 is the result of it. There was few molecular spider in a ditch. We thought that streptavidin might be spoiled.
Confirmation of forming a molecular spider
We confirmed that streptavidin can bind with biotin by electrophoresis (Fig. 8). Lane 1 is the ratio of Streptavidin to biotinylated DNA equals the ratio of 1 to 1, Lane 2 is the ratio of 1 to 2, Lane 3 is the ratio of 1 to 3, Lane 4 is the ratio of 1 to 4. The higher the ratio of biotinylated DNA, the lower electrophoretic mobility the band became. This shows that streptavidin combines with biotinylated DNA regularly.
