Biomod/2012/UTokyo/UT-Hongo/Assembly: Difference between revisions
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=Assembly of the DNA Shell= | =Assembly of the DNA Shell= | ||
First, we mixed scaffold (used base sequence of M13) and staples, and | First, we mixed scaffold (used base sequence of M13) and staples, and ascertained whether DNA Shell was hybridized as we had designed by agarose gel electrophoresis and atomic force microscope. | ||
M13 and staples were in 1X TE buffer. We prepared the solution which contain 160nM of each staple DNA and 1.6nM scaffold DNA in 1X TAE/Mg buffer and annealed it 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℃. | |||
<center> | <center> | ||
[[Image:wiki-mid.electrophoresis.jpg|frameless|500px]] | |||
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Revision as of 10:36, 15 October 2012
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<div style="height: 60px; margin-bottom: 18px;"> <div style="float: left; width: 250px; padding: 3px; background-color: white;"> <a href="http://biomod.net/"><img src="http://openwetware.org/images/8/82/Biomod2012-logo.png" width="250px" height="50px"></img></a> </div> <div style="float: right; width: 240px; padding: 3px; background-color: white;"> <a href="http://www.u-tokyo.ac.jp/en/" target="_blank"><img src="http://www.u-tokyo.ac.jp/en/images/banner/UT-logo.gif" width="234" height="60" border="0" alt="The University of Tokyo"></img></a> </div> </div>
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<li class="toppage"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo">Top</a></li> <li class="motives"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Intro">Motives</a></li> <!-- <li class="design"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Function">Design</a></li> --> <li class="result"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly">Design & Results</a> <ul class="submenu"> <li><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Assembly_of_the_DNA_Shell">Assembly of the DNA Shell</a></li> <li><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Capturing_ability">Capturing Ability</a></li> <li><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Immobilizing_on_microfluidic_device">Immobilizing on microfluidic device</a></li> <li><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Supporting_Enzyme">Supporting Enzyme</a></li> </ul> </li> <li class="method"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Method">Method</a></li> <li class="futurework"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/FutureWork">Progress & Beyond</a></li> <li class="team"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Team">Team</a></li> <li class="acknowledgement"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Acknowledgement">Acknowledgement</a></li>
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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 ascertained whether DNA Shell was hybridized as we had designed by agarose gel electrophoresis and atomic force microscope.
M13 and staples were in 1X TE buffer. We prepared the solution which contain 160nM of each staple DNA and 1.6nM scaffold DNA in 1X TAE/Mg buffer and annealed it 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
Fixing on microfluidics
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.
We made the use of this chemical reaction. Trypsin is protease. Now suppose the solution with HRP, hydrogen peroxide and TMB. In acid condition, TMB may be oxidized and change color of the solution. However, if trypsin exists with HRP solution, it can be imagined easily that trypsin decompose HRP and the chemical reaction mentioned above does not happen. Then, the DNA Shell is added. The DNA Shell protects HRP by combining streptavidin with biotin sticked to the end of spreading DNA from the Shell, so we can expect the oxidization of TMB and changing color.