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

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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

Figure 1. The check method of structure(2h anneal)

Figure 2. The check method of structure(3h anneal)

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

By means of changing annealing time,　ratio of concentration between M13 and staple, we　carried out a experiment to test influence of the time to make our structure(3D and 2D).
- About 2h annealing
  - 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).
- About 3h annealing
  - ① is only M13. ② is 2D structure. ③ is 3D structure.
  - ②and③ are M13 to staples(1:100).
  - From conclusion, 2h annealing is better than 3h, ratio of concentration(1:100) is better.

Checking the 2D and 3D structure

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

① is 100bp DNA ladder, 2.0μ.
② is cutting M13(58.5nM), 4μ+10×Loading buffer of 1μ.
③ is 2D structure(development view) sample, 10μ+10×Loading buffer of 2μ.
④ is 3D structure sample,10μ+10×Loading buffer of 2μ.

!③and④　sample data

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 for3 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 for connection development view to each other. However, if this band is connection development view to each other, it can't be seen definitely like this. This may be seen like two or more bands.

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 right, spaces labelled by:

- 1 and 5: contains 1xTA.
- 2 and 6: contains 1xTA Mg₂⁺.
- 3 and 7: contains 1xTA Zn₂⁺ (1mM).
- 4 and 8: contains 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 by the band on the right figure.

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:
Buffer: 1×TAE Mg2+
Sample:

)

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。

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

Revision as of 01:04, 29 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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@@ Line 27: / Line 27: @@
 *④ is 3D structure sample,10μ+10×Loading buffer of 2μ.
-③and④　sample data
+!③and④　sample data
 cutting M13 58.8nM      3.4μL