Biomod/2012/UTokyo/UT-Hongo/Assembly: Difference between revisions

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=Construction of the DNA Shell=
==Survey Of Results==
In this section, we write about our experiment. First, we mixed M13 and staples, and check whether DNAorigami was hybridized as designed by agarose gel electrophoresis and atomic force microscope.


==DNA Origami Assembly==
In this section, we write about our experiment.
=Assembly of the DNA Shell=


M13mp18 and staple DNA were in 1X TE buffer. The staple DNA was mixed with M13mp18 (160nM of each staple DNA, 1.6nM M13mp18, a 100-fold excess of staple DNA) in 1X TAE/Mg buffer and annealed from 95℃ to 20℃ in a themal cycler at a rate of 6.25℃ per 10minute 12 steps.
First, we mixed scaffold (used base sequence of M13) and staples, and check whether DNA origami was hybridized as we had designed by agarose gel electrophoresis and atomic force microscope.
 
 
Scaffold DNA and staples were in 1X TE buffer. The staple DNA was mixed with M13mp18 (160nM of each staple DNA, 1.6nM scaffold DNA, a 100-fold excess of staple DNA) in 1X TAE/Mg buffer and annealed from 95℃ to 20℃ in a themal cycler at a rate of 6.25℃ per 10minute 12 steps.
==Agarose Gel Electrophoresis==
==Agarose Gel Electrophoresis==


' Samples were electrophoreses in a 0.6% agarose gel containing 1xTAE/Mg buffer. The agarose gel was run at 27℃. '
'' Samples were electrophoreses in a 0.6% agarose gel containing 1xTAE/Mg buffer. The agarose gel was run at 27℃. ''
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===Outlook===
===Outlook===
First picture shows that the origami with all staples was formed as designed. Also, these pictures may show that the first origami differ from the second one in the structure between two squares.
First picture shows that the origami with all staples was formed as designed. Also, these pictures may show that the first origami differ from the second one in the structure between two squares.
=Capturing ability=
Next, we ascertained whether DNA Shell ccaptured target molecules by agarose gel electrophoresis, fluorometry, and AFM.
==Agarose gel electrophoresis==
==Fluorometry==
==AFM==
=Supporting enzyme=
Based on the results mentioned above, we did further advanced experiment. We ascertained whether DNA Shell supported enzymes.
==Background==
We used tetramethylbenzidine (TMB), streptavidin with horseradish peroxidase (HRP) labeling and trypsin. TMB can be oxidized for the reduction of hydrogen peroxide to water by peroxidase enzymes such as HRP. When TMB is oxidized, the color of the solution takes on a blue color. If pH of the solution is low (for example, using sulfric acid), the color turns into yellow. The former blue color can be read at a wavelength of nm and the latter yellow color can be read at nm.
==Experiment condition==

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In this section, we write about our experiment.

Assembly of the DNA Shell

First, we mixed scaffold (used base sequence of M13) and staples, and check whether DNA origami was hybridized as we had designed by agarose gel electrophoresis and atomic force microscope.


Scaffold DNA and staples were in 1X TE buffer. The staple DNA was mixed with M13mp18 (160nM of each staple DNA, 1.6nM scaffold DNA, a 100-fold excess of staple DNA) in 1X TAE/Mg buffer and annealed from 95℃ to 20℃ in a themal cycler at a rate of 6.25℃ per 10minute 12 steps.

Agarose Gel Electrophoresis

Samples were electrophoreses in a 0.6% agarose gel containing 1xTAE/Mg buffer. The agarose gel was run at 27℃.

The band of M13+staple ran longer than the band of M13, it showed that the structure which had big molecular mass was created. The lower fuzzy band was the band of excess staple DNA. RB and LB means Right Bridge and Left Bridge. These connect three squares of DNA origami.

AFM

These DNA origami (M13 + all staples, M13 + all staples except ones which combine with M13 between the squares (except LB and RB), M13 + all staples except one which combine with M13 between the center square and the right square (except RB)) were observed by using atomic force microscope (AFM) to confirm that these DNA origami were formed correctly. 1xTAE/Mg solution was utilized as buffer.

M13+staple (represent 500nm at one side)



M13+staple (without LB,RB)



M13+staple (without RB)

Now trying..

Outlook

First picture shows that the origami with all staples was formed as designed. Also, these pictures may show that the first origami differ from the second one in the structure between two squares.


Capturing ability

Next, we ascertained whether DNA Shell ccaptured target molecules by agarose gel electrophoresis, fluorometry, and AFM.

Agarose gel electrophoresis

Fluorometry

AFM

Supporting enzyme

Based on the results mentioned above, we did further advanced experiment. We ascertained whether DNA Shell supported enzymes.

Background

We used tetramethylbenzidine (TMB), streptavidin with horseradish peroxidase (HRP) labeling and trypsin. TMB can be oxidized for the reduction of hydrogen peroxide to water by peroxidase enzymes such as HRP. When TMB is oxidized, the color of the solution takes on a blue color. If pH of the solution is low (for example, using sulfric acid), the color turns into yellow. The former blue color can be read at a wavelength of nm and the latter yellow color can be read at nm.

Experiment condition

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