Biomod/2011/TeamJapan/Sendai/Notes: Difference between revisions

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To improve the efficiency of the molecular robot we gathered many ideas related with the robot design and its mechanism for movement:
To improve the efficiency of the molecular robot we gathered many ideas related with the robot design and its mechanism for movement:


=== Hexagonal prism (suspended) ===
=== Hexagonal prism (suspended) ===
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''Advantage''<br />
''Advantage''<br />
This robot will always go towards one direction.<br />
This robot will always go towards one direction. <br />
A novel mechanism for motion.<br />
A novel mechanism for motion.<br />


''Disadvantage''<br />
''Disadvantage''<br />
This robot may fall at the time of observation by AFM.<br />
This robot may fall at the time of observation by AFM. <br />
The difficulty to make a single wall.<br />
The difficulty to make a single wall.<br />


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In addition the legs design include both kind of robots for saving time and costs.<br />
In addition the legs design include both kind of robots for saving time and costs.<br />
[http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Notes/Stick '''Here'''] you can find this robot sequence and more information.
[http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Notes/Stick '''Here'''] you can find this robot sequence and more information.


== Our activity progress ==
== Our activity progress ==
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=== The hexagonal prism ===
=== The hexagonal prism ===
Annealing of robot’s body<br />
Annealing of robot’s body<br />
Electrophoresis<br />
Electrophoresis


=== The triangular prism ===
=== The triangular prism ===
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Cutting of leg and substrate with spider<br />
Cutting of leg and substrate with spider<br />
Putting robot on field<br />
Putting robot on field<br />


== The methods used for the experiments  ==
== The methods used for the experiments  ==
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Gel: 24% Poly-Acrylamide Gel<br />
Gel: 24% Poly-Acrylamide Gel<br />
Buffer: 1×TAE Mg<sup>2+</sup><br />
Buffer: 1×TAE Mg<sup>2+</sup><br />
Leg and substrate with fluorescence were diluted to 100nM, and they were taken every 22 micro / L. As a buffer, 1xTA Mg<sup>2+</sup> and 1xTA Mg<sup>2+</sup> with Zn<sup>2+</sup> (1mM, 2mM, 10mM) were put in. Time after putting in buffer was set to 0min, 5min, and 30min. As a buffer for electrophoresis, 1xTA Mg<sup>2+</sup> was used. As a result of electrophoresis, it did not turn out what the bands would show, since only substrate and only leg were not put in as sample. Moreover, there is no telling whether leg and substrate have constructed double helix correctly at normal temperature. So such things were taken into consideration in the next electrophoresis.  
Leg and substrate with fluorescence were diluted to 100nM, and they were taken every 22 micro / L. As a buffer, 1xTA Mg<sup>2+</sup> and 1xTA Mg<sup>2+</sup> with Zn<sup>2+</sup> (1mM, 2mM, 10mM) were put in. Time after putting in buffer was set to 0min, 5min, and 30min. As a buffer for electrophoresis, 1xTA Mg<sup>2+</sup> was used. As a result of electrophoresis, it did not turn out what the bands would show, since only substrate and only leg were not put in as sample. Moreover, there is no telling whether leg and substrate have constructed double helix correctly at normal temperature. So such things were taken into consideration in the next electrophoresis.  
{{-}}
{{-}}


[[Image:Leg cut 2kaime.JPG|320px|thumb|Figure3 .Second experiment. 1; only legs 2; only substrates 3, 5, 7 and 9; 1×TAE Mg<sub>2</sub><sup>+</sup>　4, 6, 8, 10 and 11; 1×TAE Mg<sub>2</sub><sup>+</sup> with Zn<sub>2</sub><sup>+</sup> 1mM. 3 and 4; 0min after adding buffer. 5 and 6; 15min after.　7, 8; 15min after (heat-treated).　9 and 10; 15min after (1/3 concentration).　11; 15min after (heat-treated, 1/3 concentration).]]
[[Image:Leg cut 2kaime.JPG|320px|thumb|Figure3 .Second experiment. 1; only legs. 2; only substrates. 3, 5, 7 and 9; 1×TAE Mg<sub>2</sub><sup>+</sup>.　46810111×TAE Mg<sub>2</sub><sup>+</sup> with Zn<sub>2</sub><sup>+</sup> 1mM.  
340min after adding buffer. 5615min after　7815min after (heat-treated).　91015min after (1/3 concentration).　1115min after (heat-treated, 1/3 concentration).]]
 


2.Experiment conditions:<br />
2.Experiment conditions:<br />
Gel:24% Poly-Acrylamide Gel<br />
Gel:24% Poly-Acrylamide Gel<br />
Buffer:1×TAE Mg<sup>2+</sup><br />
Buffer:1×TAE Mg<sup>2+</sup><br />
As a new sample, sample-A and sample-B were added. Sample-A : The substrate and leg are diluted to 100nM and they are put in every 22micro / L . After dipping in boiling water for 2~3 minutes, it dips in cold water for 10 minutes. Buffer is put in after making 2 chains construct certainly. Sample-B : Concentration is set to one third. Because it thought that substrate and leg which were separated approached closely since concentration was too dense, and fluorescence can not react. As a result of electrophoresis, cutting can be checked in all the samples. Therefore, it is thought that substrate and leg will be cut by acting of Mg<sub>2</sub><sup>+</sup> even if Zn<sub>2</sub><sup>+</sup> is not included. Moreover, even if heat treatment did not added, the double helix was constructed exactly, and even if it does not make concentration thin, cutting has fully checked. It is considered as a cause that Mg<sub>2</sub><sup>+</sup> is acting. In the next experiment, the effect of Mg<sub>2</sub><sup>+</sup> was observed by using buffer which does not contain Mg<sub>2</sub><sup>+</sup> for gel and electrophoresis.  
As a new sample, sample-A and sample-B were added. Sample-A : The substrate and leg are diluted to 100nM and they are put in every 22micro / L . After dipping in boiling water for 2~3 minutes, it dips in cold water for 10 minutes. Buffer is put in after making 2 chains construct certainly. Sample-B : Concentration is set to one third. Because it thought that substrate and leg which were separated approached closely since concentration was too dense, and fluorescence can not react. As a result of electrophoresis, cutting can be checked in all the samples. Therefore, it is thought that substrate and leg will be cut by acting of Mg<sub>2</sub><sup>+</sup> even if Zn<sub>2</sub><sup>+</sup> is not included. Moreover, even if heat treatment did not added, the double helix was constructed exactly, and even if it does not make concentration thin, cutting has fully checked. It is considered as a cause that Mg<sub>2</sub><sup>+</sup> is acting. In the next experiment, the effect of Mg<sub>2</sub><sup>+</sup> was observed by using buffer which does not contain Mg<sub>2</sub><sup>+</sup> for gel and electrophoresis.  
{{-}}
 


3.By putting in buffer containing Mg<sub>2</sub><sup>+</sup>, legs and substrate were cut (refer to [http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis#DNAzyme_Legs_and_a_substrate_with_internal_ribonucleotide_and_fluorescence_protein_at_5.E2.80.99_end this page]). As an action of Zn2+, it turned out from the difference of luminosity in 0min that the initial velocity of cutting becomes quick a little by using buffer containing Zn<sub>2</sub><sup>+</sup>.  
3.By putting in buffer containing Mg<sub>2</sub><sup>+</sup>, legs and substrate were cut (refer to [http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis#DNAzyme_Legs_and_a_substrate_with_internal_ribonucleotide_and_fluorescence_protein_at_5.E2.80.99_end this page]). As an action of Zn2+, it turned out from the difference of luminosity in 0min that the initial velocity of cutting becomes quick a little by using buffer containing Zn<sub>2</sub><sup>+</sup>.  


==== Substrate without fluorescence ====
==== Substrate without fluorescence ====
Since a stain solution was not used in the experiment using substrate with fluorescence, in order to investigate whether it can be checked that reg and substrate have been cut even when a stain solution is used, carried out experiment using substrate without fluorescence.
Since a stain solution was not used in the experiment using substrate with fluorescence, in order to investigate whether it can be checked that reg and substrate have been cut even when a stain solution is used, carried out experiment using substrate without fluorescence.




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==== '''Form a double helix''' ====
==== '''Form a double helix''' ====
Mix 5μL M13(84nM), 3μL DNA that specific part of m13 sequence form a double helix(5μM), 2μL Buffer BSA, 2μL BalⅠ buffer, and 5μL mQ.
Mix 5μL M13(84nM), 3μL DNA that specific part of m13 sequence form a double helix(5μM), 2μL Buffer BSA, 2μL BalⅠ buffer, and 5μL mQ.<br />
Set in PCR, the condition is 3min at 95°C, 3min at 65°C, for the duration of enzyme reaction, let the sample at 37°C.
Set in PCR, the condition is 3min at 95°C, 3min at 65°C, for the duration of enzyme reaction, let the sample at 37°C.<br />


==== '''Cutting M13 by enzyme reaction''' ====
==== '''Cutting M13 by enzyme reaction''' ====
Add 1μL BalⅠin the sample, let the sample stand for 2 hours at 37°C.<br />
Add 1μL BalⅠin the sample, let the sample stand for 2 hours at 37°C.<br />
Add 3μL 10×H buffer and 7μL mQ, add 1μL PstⅠ, let the sample stand for 1 hours at 37°C.<br />
Add 3μL 10×H buffer and 7μL mQ, add 1μL PstⅠ, let the sample stand for 1 hours at 37°C.<br />


==== '''Clean up of enzyme''' ====
==== '''Clean up of enzyme''' ====
:'''The first method'''
'''The first method'''
:*All centrifugation steps are carried out at 16,000×g(13,000 rpm) in a conventional tabletop micro-centrifuge at room temperature.
*All centrifugation steps are carried out at 16,000×g(13,000 rpm) in a conventional tabletop micro-centrifuge at room temperature.<br />


:Add 5 volumes of Buffer PBI to 1 volume of the cutting M13 and mix.<br />
Add 5 volumes of Buffer PBI to 1 volume of the cutting M13 and mix.<br />
:Place a columns in a 2ml collection tube.<br />
Place a columns in a 2ml collection tube.<br />
:To bind DNA, apply the sample to the columns and centrifuge for 1min.<br />
To bind DNA, apply the sample to the columns and centrifuge for 1min.<br />
:Discard flow-through.Place the columns back into the same tube.<br />
Discard flow-through.Place the columns back into the same tube.<br />
:To wash, add 0.75ml Buffer PE to the columns and centrifuge for 1min.<br />
To wash, add 0.75ml Buffer PE to the columns and centrifuge for 1min.<br />
:Discard flow-through and place the columns back in the same tube. Centrifuge the columns for additional 1 min.<br />
Discard flow-through and place the columns back in the same tube. Centrifuge the columns for additional 1 min.<br />
:Place columns in a clean 1.5ml micro-centrifuge tube.<br />
Place columns in a clean 1.5ml micro-centrifuge tube.<br />
:To elute DNA, add 25 μL Buffer EB (10mM Tris-Cl, pH8.5) to the center of the columns, let the columns stand for 1 min, and then centrifuge for 1 min.<br />
To elute DNA, add 25 μL Buffer EB (10mM Tris-Cl, pH8.5) to the center of the columns, let the columns stand for 1 min, and then centrifuge for 1 min.<br />


:The first method is to make sure of cut M13, so this method is mixing long cut M13 and short(we want) cut M13.
The first method is to make sure of cut M13, so this method is mixing long cut M13 and short(we want) cut M13.<br />


:'''The second method'''<br />
:*All centrifugation steps are carried out at 16,000×g(13,000 rpm) in a conventional tabletop micro-centrifuge at room temperature.


:Add 1/10 volumes of Sodium Acetate, and 2.5 volumes of ethanol to 1 volume of the cutting M13 and mix, and then centrifuge for 10 min.
'''The second method'''<br />
:Discard supernatant being careful not to throw out DNA pellet.
 
:Dissolve pellet in 1×TE buffer.
*All centrifugation steps are carried out at 16,000×g(13,000 rpm) in a conventional tabletop micro-centrifuge at room temperature.<br />
:After electrophoresis, excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
 
:Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
Add 1/10 volumes of Sodium Acetate, and 2.5 volumes of ethanol to 1 volume of the cutting M13 and mix, and then centrifuge for 10 min.<br />
:Weight the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel.
Discard supernatant being careful not to throw out DNA pellet.<br />
:Incubate at 50°C for 10 min. To help dissolve gel, mix by vortexing the tube every 3 min during the incubation.
Dissolve pellet in 1×TE buffer.<br />
:After the gel slice has dissolve completely, add 1 gel volumes of isopropanol to the sample and mix.
After electrophoresis, excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
:Place a columns in a provided 2ml collection tube.
Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.<br />
:To bind DNA, apply the sample to the columns, and centrifuge for 1 min.
Weight the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel.<br />
:Discard flow-though and place columns back in the same collection tube.
Incubate at 50°C for 10 min. To help dissolve gel, mix by vortexing the tube every 3 min during the incubation.<br />
:Add 0.5ml of Buffer QG to columns and centrifuge for 1 min.
After the gel slice has dissolve completely, add 1 gel volumes of isopropanol to the sample and mix.<br />
:To wash, add 0.75 ml of Buffer PE to columns and centrifuge for 1 min.
Place a columns in a provided 2ml collection tube.<br />
:Discard the flow-though and centrifuge the columns for an additional 1 min.
To bind DNA, apply the sample to the columns, and centrifuge for 1 min.<br />
:Place columns into a clean 1.5 ml micro-centrifuge tube.
Discard flow-though and place columns back in the same collection tube.<br />
:To elute DNA, add 25 μL Buffer EB (10mM Tris-Cl, pH8.5) to the center of the columns, let the columns stand for 1 min, and then centrifuge for 1 min.
Add 0.5ml of Buffer QG to columns and centrifuge for 1 min.<br />
To wash, add 0.75 ml of Buffer PE to columns and centrifuge for 1 min.<br />
Discard the flow-though and centrifuge the columns for an additional 1 min.<br />
Place columns into a clean 1.5 ml micro-centrifuge tube.<br />
To elute DNA, add 25 μL Buffer EB (10mM Tris-Cl, pH8.5) to the center of the columns, let the columns stand for 1 min, and then centrifuge for 1 min.<br />
 
The second method can purify only short(we want) M13.<br />
 


:The second method can purify only short(we want) M13.


==  Molecular spider's experiment as practice==
==  Molecular spider's experiment as practice==
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We guess that our failure was resulted from that our spider was not purified　through HPLC.
We guess that our failure was resulted from that our spider was not purified　through HPLC.
{{-}}
{{-}}


== DNA sequence ==
== DNA sequence ==

Revision as of 00:30, 2 November 2011

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<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> 

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Brainstorming

Figure 1.Brainstorming

To improve the efficiency of the molecular robot we gathered many ideas related with the robot design and its mechanism for movement:


Hexagonal prism (suspended)

By design the motion of this kind of robot is rolling rather than walking. This robbot was our first plan.

Advantage
A maximum of 72 legs can be attached to a hexagonal prismatic body.
The robot does not go backwards. Therefore, walking forward efficiently.

Disadvantage
The difficulty in verifying the 3D body design.
The time cost that is necessary to get the robot annealed.
The complexity of the structure due to the different variety of staples.

Here you can find this robot sequence and more information.


Wall runner (suspended)

Over the path we can create two walls that link the start point and goal, analogous to a simple labyrinth.

Advantage
This robot will always go towards one direction.
A novel mechanism for motion.

Disadvantage
This robot may fall at the time of observation by AFM.
The difficulty to make a single wall.


Sticky motif robot (suspended)

This design is a very simple one, where it is combined only four DNA double strands.
The sticky motif robot was our plan B in case we failed to produce the triangular prism robot.
In addition the legs design include both kind of robots for saving time and costs.
Here you can find this robot sequence and more information.


Our activity progress

Here is stage at where our respective designs are:

The hexagonal prism

Annealing of robot’s body
Electrophoresis

The triangular prism

Annealing of robot’s body
Electrophoresis
Annealing of robot’s body and legs
Cutting of excessive M13

Legs

Annealing of field
Cutting of leg and substrate with spider
Putting robot on field


The methods used for the experiments

Freeze'n squeeze

This experiment refers to Harvard team wiki.
Thank you so much Harvard team!!

DNAzyme legs cutting substrates

Substrate with fluorescence

Leg and substrate type A, which attached fluorescence and quencher, have constructed the double helix. By putting in buffer containing Zn2+, fluorescence is separated from quencher, and fluoresce. Therefore, by observing fluorescence, it can be confirmed whether legs and substrate were cut. The process is shown below.

Figure 2. First experiment. All samples include both legs and substrates. Buffer:1; 1×TAE Mg2+. 2, 5, and 8; 1×TAE Mg2+ with Zn2+1mM. 3, 6 and 9; 1×TAE Mg2+ with Zn2+2mM. 4, 7 and 10; 1×TAE Mg2+ with Zn2+10mM. Time after adding buffer:2, 3 and 4; 0min. 5, 6 and 7; 5min. 8, 9 and 10; 30min.

1.Experiment conditions:
Gel: 24% Poly-Acrylamide Gel
Buffer: 1×TAE Mg2+

Leg and substrate with fluorescence were diluted to 100nM, and they were taken every 22 micro / L. As a buffer, 1xTA Mg2+ and 1xTA Mg2+ with Zn2+ (1mM, 2mM, 10mM) were put in. Time after putting in buffer was set to 0min, 5min, and 30min. As a buffer for electrophoresis, 1xTA Mg2+ was used. As a result of electrophoresis, it did not turn out what the bands would show, since only substrate and only leg were not put in as sample. Moreover, there is no telling whether leg and substrate have constructed double helix correctly at normal temperature. So such things were taken into consideration in the next electrophoresis.

Figure3 .Second experiment. 1; only legs. 2; only substrates. 3, 5, 7 and 9; 1×TAE Mg2+.　46810111×TAE Mg2+ with Zn2+ 1mM. 340min after adding buffer. 5615min after　7815min after (heat-treated).　91015min after (1/3 concentration).　1115min after (heat-treated, 1/3 concentration).


2.Experiment conditions:
Gel:24% Poly-Acrylamide Gel
Buffer:1×TAE Mg2+

As a new sample, sample-A and sample-B were added. Sample-A : The substrate and leg are diluted to 100nM and they are put in every 22micro / L . After dipping in boiling water for 2~3 minutes, it dips in cold water for 10 minutes. Buffer is put in after making 2 chains construct certainly. Sample-B : Concentration is set to one third. Because it thought that substrate and leg which were separated approached closely since concentration was too dense, and fluorescence can not react. As a result of electrophoresis, cutting can be checked in all the samples. Therefore, it is thought that substrate and leg will be cut by acting of Mg2+ even if Zn2+ is not included. Moreover, even if heat treatment did not added, the double helix was constructed exactly, and even if it does not make concentration thin, cutting has fully checked. It is considered as a cause that Mg2+ is acting. In the next experiment, the effect of Mg2+ was observed by using buffer which does not contain Mg2+ for gel and electrophoresis.


3.By putting in buffer containing Mg2+, legs and substrate were cut (refer to this page). As an action of Zn2+, it turned out from the difference of luminosity in 0min that the initial velocity of cutting becomes quick a little by using buffer containing Zn2+.

Substrate without fluorescence

Since a stain solution was not used in the experiment using substrate with fluorescence, in order to investigate whether it can be checked that reg and substrate have been cut even when a stain solution is used, carried out experiment using substrate without fluorescence.


Method for cutting the M13

To cut m13, specific part of m13 sequence form a double helix, secondly react by enzyme, finally clean up of discarded enzyme and pick out only M13. The process is shown below.

Form a double helix

Mix 5μL M13(84nM), 3μL DNA that specific part of m13 sequence form a double helix(5μM), 2μL Buffer BSA, 2μL BalⅠ buffer, and 5μL mQ.
Set in PCR, the condition is 3min at 95°C, 3min at 65°C, for the duration of enzyme reaction, let the sample at 37°C.

Cutting M13 by enzyme reaction

Add 1μL BalⅠin the sample, let the sample stand for 2 hours at 37°C.
Add 3μL 10×H buffer and 7μL mQ, add 1μL PstⅠ, let the sample stand for 1 hours at 37°C.


Clean up of enzyme

The first method

  • All centrifugation steps are carried out at 16,000×g(13,000 rpm) in a conventional tabletop micro-centrifuge at room temperature.

Add 5 volumes of Buffer PBI to 1 volume of the cutting M13 and mix.
Place a columns in a 2ml collection tube.
To bind DNA, apply the sample to the columns and centrifuge for 1min.
Discard flow-through.Place the columns back into the same tube.
To wash, add 0.75ml Buffer PE to the columns and centrifuge for 1min.
Discard flow-through and place the columns back in the same tube. Centrifuge the columns for additional 1 min.
Place columns in a clean 1.5ml micro-centrifuge tube.
To elute DNA, add 25 μL Buffer EB (10mM Tris-Cl, pH8.5) to the center of the columns, let the columns stand for 1 min, and then centrifuge for 1 min.

The first method is to make sure of cut M13, so this method is mixing long cut M13 and short(we want) cut M13.


The second method

  • All centrifugation steps are carried out at 16,000×g(13,000 rpm) in a conventional tabletop micro-centrifuge at room temperature.

Add 1/10 volumes of Sodium Acetate, and 2.5 volumes of ethanol to 1 volume of the cutting M13 and mix, and then centrifuge for 10 min.
Discard supernatant being careful not to throw out DNA pellet.
Dissolve pellet in 1×TE buffer.
After electrophoresis, excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
Weight the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel.
Incubate at 50°C for 10 min. To help dissolve gel, mix by vortexing the tube every 3 min during the incubation.
After the gel slice has dissolve completely, add 1 gel volumes of isopropanol to the sample and mix.
Place a columns in a provided 2ml collection tube.
To bind DNA, apply the sample to the columns, and centrifuge for 1 min.
Discard flow-though and place columns back in the same collection tube.
Add 0.5ml of Buffer QG to columns and centrifuge for 1 min.
To wash, add 0.75 ml of Buffer PE to columns and centrifuge for 1 min.
Discard the flow-though and centrifuge the columns for an additional 1 min.
Place columns into a clean 1.5 ml micro-centrifuge tube.
To elute DNA, add 25 μL Buffer EB (10mM Tris-Cl, pH8.5) to the center of the columns, let the columns stand for 1 min, and then centrifuge for 1 min.

The second method can purify only short(we want) M13.


Molecular spider's experiment as practice

For practice to observe by AFM, we carried out experiment about molecular spider.

  • The creating method of STV:C-Leg=1:4

STV 5mg/ml in K2HPO4:KH2PO4=5:11 pH6.5　2.1μl
Capture-Leg 16nM 8μl
Mg2+ 1M 0.6μ
Tris:Tris-HCl=2.5:7.4 pH7.4 1μ
MQ 38.3μl
In this case,all is 50μl.

We failed to observe the molecular spider on DNA origami field. We guess that our failure was resulted from that our spider was not purified　through HPLC.


DNA sequence

You can download the staples DNA sequences for the triangular prism body and the DNA origami field by clicking here.

Figure5. DNA origami Field
Figure6: triangular prism sequence of staples.
red for leg, green for prism, purple for capture-leg
Figure7. Field & substrate & leg

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