Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis: Difference between revisions

Revision as of 04:34, 30 October 2011

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 <td>

<ul id="verticalmenu" class="glossymenu"> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai">Home</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Strategy">Strategy</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Design">Design</a></li> <li><a href="#">Experiments</a>

   <ul>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis">Electrophoresis</a> 
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Atomic_Force_Microscope">AFM</a> 
   </ul>

</li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Computational_design/Simulation" >Simulation</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Notes">Notes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Team">Team</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Resources">Resources</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Sitemap">Sitemap</a></li>

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Method for checking the structure

triangular prism
- gel:1.4% agarose gel
- buffer:1×TAE Mg2+

By means of changing annealing time　and　ratio of concentration between M13 and staple, we　carried out a experiment to test influence of the time and ratio to make our structure(3D and 2D).
From conclusion, 2h annealing is better than 3h, ratio of concentration(1:100) is better.

Figure 1. The check method of structure(2h anneal) ① is only M13. ②and④ are 2D structure. ③and⑤ are 3D structure. ②and③ are M13 to staples(1:10). ④and⑤ are M13 to staples(1:100).

Figure 2. The check method of structure(3h anneal) ① is only M13. ② is 2D structure. ③ is 3D structure.②and③ are M13 to staples(1:100).

Checking the 2D and 3D structure

We carried out electrophoresis　to 2D and 3D structure to check the differences among two. Figure3 shows between 2D and 3D structure's band gap.

③and④　sample data
	x μL
cutting M13 58.8nM	3.4μL
structure staples 400nM	5μL
5×TAE-Mg²⁺	10μL
Distiled Water	31.6μL
Total	50μL

Annealing time 95°C→25°C -1°C for 3 minute

Band C shows cutting M13. Band B is 2D structure, because this band is up on M13's band. Band A may be 3D structure's band.　Two bands have appeared in the lane of 3D sample. One of them is in the same height as the band of the next 2D. There are two possibilities of thinking here. One is a band of triangular prism structure. Another is the band which is made by connection of development views to each other. However, if this band is connection of development views to each other, it can't be seen definitely like this. It may be seen like two or more bands. This is why we judged experiment of making 3D structure was success. As correct the proof of this calculation, please see this page.

Method for checking the cutting legs and substrates

Legs and substrate with attached fluorescence protein

Gel: 24% Poly-Acrylamide Gel
Buffer: 1×TAE
Sample: Legs and substrates are contained in the sample.

As shown on the figure at the below, wells labelled by:

- 1 and 5 contain 1xTA.
- 2 and 6 contain 1xTA Mg₂⁺.
- 3 and 7 contain 1xTA Zn₂⁺ (1mM).
- 4 and 8 contain 1xTA Mg₂⁺ Zn₂⁺ (1mM).

Where 1, 2, 3 and 4 are immediately placed after solubilization in buffer, but 5, 6, 7 and 8 are placed 15min later after solubilization in buffer. As time passes, it results that the quantity of cut substrates increases because it can be checked that substrates at 2, 4, 6 and 8 are cut. Figure 4より0minでのバンドの輝度差からZn2+を入れる事で倍近く切れる量が変わっていることがわかる

Legs and substrate unattached fluorescence protein

Gel: 24% Poly-Acrylamide Gel
Buffer: 1×TAE
Sample: Legs and substrates are contained in 1, 2, 3 and 4. Only substrates and legs are contained in 5 and 6, respectively.

Moreover, 1xTE Mg2+ Zn2+ (1mM) is contained in 1 as buffer. Similarly, 1xTE Zn2+ (1mM) is contained in 2, 1xTE Mg2+ is contained in 3, and 1xTE is contained in 4, 5 and 6. Where all the samples are required to be have been solubilized in a buffer for 15min. From the band of the right figure, we can check that substrates of 1, 2 and 3 are cut. This is due to the fact that the double helix was constructed using a thermal cycler as a reason which has checked cutting in 2 this time.

Micro spin calm

(Micro spin calmを用いて余ったstapleを除去した。その際構造体にどのような影響があるかを電気泳動により観察した。この方法では電気泳動の結果からは余ったstapleが除去できかつ構造体のバンドが見えているため良いのだがAFMでは何も観察できていないことから構造体が壊れてしまったと考えられる。

Gel: 0.7% agaros
Buffer: 1×TAE Mg2+
Sample:

100V 90minute ) We observed with electrophoresis the influence which removing the surplus leg with micro spin calm　has on structure.

Method for cutting M13

File:M13cut for wiki.jpg

Figure 6. Cutting M13

Gel: 1.0% agarose gel
Buffer: 1×TAE
Sample: ①は無切断M13で、②は切断したM13

私たちのロボットはscaffoldとしてM13（7,249bases）を用いており、その中の1,108basesを使用する。M13をそのまま用いて構造体を作るとM13の大部分が余ってしまい、AFMでの構造体の判別が困難になる。そのため、私たちは制限酵素を用いてM13を切断する実験を行った。図の6は切断したものとそのままのM13を電気泳動で比較したものである。レーン①は無切断M13で、②切断したM13また、AはM13のバンドであり、BはM13が切断された不要な長い方のバンドであり、CはM13が切断された求めていた短い方のバンドであり、DはM13を切断するために二重螺旋を組ませる短いDNA(20base)の余りのバンドである。なお切断したM13はnoteに書いた方法で精製したものである。

For producing the robot body we used the viral M13mp18 DNA single strand (M13) as scaffold. But we only used 1,108 bases of 7,249 bases , then having a leftover. In this situation we thought about cutting the part of M13 that we needed. Our first attempt was to extract the necessary part of M13 to reproduce M13 with polymerase chain reaction. But we failed. So we changed our method for cutting the M13 with restriction enzyme and did experiment. In figure 6, M13 was compared whe bnith cutting M13 using electrophoresis. ① is M13 and ② is cutting M13. A is the band of M13. B is the band of M13 of redundunt. C is the band of the part of M13 that we needed. D is the band of the remainder of short DNA(20base) which is combined with M13 to cut M13. Cutting M13 is produced the way on NOTES of our wiki.

@@ Line 69: / Line 69: @@
 *Buffer: 1×TAE
 *Sample: Legs and substrates are contained in the sample.
-As shown on the figure at the right, spaces labelled by:
+As shown on the figure at the below, wells labelled by:
-**1 and 5: contains 1xTA.
+**1 and 5 contain 1xTA.
-**2 and 6: contains 1xTA Mg<sub>2</sub><sup>+</sup>.
+**2 and 6 contain 1xTA Mg<sub>2</sub><sup>+</sup>.
-**3 and 7: contains 1xTA Zn<sub>2</sub><sup>+</sup> (1mM).
+**3 and 7 contain 1xTA Zn<sub>2</sub><sup>+</sup> (1mM).
-**4 and 8: contains 1xTA Mg<sub>2</sub><sup>+</sup> Zn<sub>2</sub><sup>+</sup> (1mM).
+**4 and 8 contain 1xTA Mg<sub>2</sub><sup>+</sup> Zn<sub>2</sub><sup>+</sup> (1mM).
 Where 1, 2, 3 and 4 are immediately placed after solubilization in buffer, but 5, 6, 7 and 8 are placed 15min later after solubilization in buffer.
-As time passes, it results that the quantity of cut substrates increases because it can be checked that substrates at 2, 4, 6 and 8 are cut by the band on the right figure.
+As time passes, it results that the quantity of cut substrates increases because it can be checked that substrates at 2, 4, 6 and 8 are cut.
 Figure 4より0minでのバンドの輝度差からZn2+を入れる事で倍近く切れる量が変わっていることがわかる
 {|align=center

Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis: Difference between revisions

Revision as of 04:34, 30 October 2011

Contents

Method for checking the structure

Checking the 2D and 3D structure

Method for checking the cutting legs and substrates

Legs and substrate with attached fluorescence protein

Legs and substrate unattached fluorescence protein

Micro spin calm

Method for cutting M13

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