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

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{{:Biomod/2011/TeamJapan/Sendai/HeaderElectro}}
{{:Biomod/2011/TeamJapan/Sendai/HeaderElectro}}
== About the methods ==
In this section we are presenting the procedures we took for the following cases:
*Verifying [http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Design#Designing_the_molecular_robot 2D and 3D structures]. Basically we verified whether the folding was properly done or not.
*Verifying whether the [http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Design#Leg_Design DNAzyme legs cut the substrates]. Important step for the robot motion.
*After the folding, there were some [http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Design#Verification_of_our_structure_and_field_from_the_mix_sample leftover staples]. We needed to get rid of them before the next step.
*Because we [http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Design#Procedure_for_cutting_DNA cut M13] it is needed to check whether we got the desire 1,108 nucleotides.
Take into consideration that DNA strands were stained by SYBR Gold (sigma) except when observing them with fluorescence.


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<A Href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis#Method_for_checking_the_structure">
<A Href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis#Method_for_verifying_the_structure">
<Img Src="http://openwetware.org/images/c/cc/Structur.png" Border="0" Width="400" Height="150"><br>Method for checking the structure</a>
<Img Src="http://openwetware.org/images/c/cc/Structur.png" Border="0" Width="400" Height="150"><br>Method for verifying the structure</a>
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<A Href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis#Method_for_checking_DNAzyme_legs_cut_substrates">
<A Href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis#Method_for_verifying_that_the_DNAzyme_legs_cut_the_substrates">
<Img Src="http://openwetware.org/images/1/1c/Leg-cut.png" Border="0" Width="400" Height="150"　align=""right""><br>Method for checking DNAzyme legs cut substrates</a>
<Img Src="http://openwetware.org/images/1/1c/Leg-cut.png" Border="0" Width="400" Height="150"　align=""right""><br>Method for verifying that the DNAzyme legs cut substrates</a>
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== Notice ==
In this electrophoresis section, DNA were stained by SYBR Gold (sigma) except observing DNA with fluoresce.


== Method for verifying the structure  ==
== Method for verifying the structure  ==
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Annealing:  from 90ºC to 25 ºC (decreasing 1 ºC each 3 minutes)
Annealing:  from 90ºC to 25 ºC (decreasing 1 ºC each 3 minutes)


As a result of the electrophoresis: Band C indicates the cut M13. Band B indicates the 2D structure, because this band shows an slower migration than cut M13. Two bands were observed in line 4, and the lower band showed similar migration as the band in line 3. There are two possibilities to consider in case of the upper band. First, one of the two bands indicates the band of the triangular prism structure. Second, this is the band which was made by connection of 2D structures between each other. However, if the upper band indicated connection of two pieces of 2D structures, two or more bands should have been observed. Thus, we arrived to the conclusion that the upper band indicates the designed 3D structure. Our AFM observation supports this conclusion. Please see [http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Atomic_Force_Microscope#Observation_of_2D_and_3D_structures this page] for the AFM observation.
As a result of the electrophoresis: Band C indicates the cut M13. Band B indicates the 2D structure, because this band shows an slower migration than cut M13. Two bands were observed in lane #4, and the lower band showed similar migration as the band in lane #3. There are two possibilities to consider in case of the upper band. First, one of the two bands indicates the band of the triangular prism structure. Second, this is the band which was made by connection of 2D structures between each other. However, if the upper band indicated connection of two pieces of 2D structures, two or more bands should have been observed. Thus, we arrived to the conclusion that the upper band indicates the designed 3D structure. Our AFM observation supports this conclusion. Please see [http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Atomic_Force_Microscope#Observation_of_2D_and_3D_structures this page] for the AFM observation.
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== Method for checking DNAzyme legs cut substrates  ==
== Method for verifying that the DNAzyme legs cut the substrates  ==
=== DNAzyme Legs and a substrate with internal ribonucleotide and fluorescence protein at 5’ end ===
=== DNAzyme Legs and a substrate with internal ribonucleotide and fluorescence protein at 5’ end ===
Experiment conditions:
Experiment conditions:
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In figure 3, samples loaded in each well are:
In figure 3, samples loaded in each well are:
1 and 5 contain 1xTA<br />
1 and 5 contain 1xTA<br />
2 and 6 contain 1xTA Mg<sub>2</sub><sup>+</sup>(12.5mM)<br />
2 and 6 contain 1xTA Mg<sub>2</sub><sup>+</sup> (12.5mM)<br />
3 and 7 contain 1xTA Zn<sub>2</sub><sup>+</sup> (1mM)<br />
3 and 7 contain 1xTA Zn<sub>2</sub><sup>+</sup> (1mM)<br />
4 and 8 contain 1xTA Mg<sub>2</sub><sup>+</sup>(12.5mM) and Zn<sub>2</sub><sup>+</sup> (1mM)<br />
4 and 8 contain 1xTA Mg<sub>2</sub><sup>+</sup> (12.5mM) and Zn<sub>2</sub><sup>+</sup> (1mM)<br />
Where 1-4 are immediately loaded after mixing, and 5-8 are loaded after incubation during 15 min at room temperature. Electrophoresis showed bands which migrated faster than original substrates. The faste migrated bands indicate cleaved substrate, and that were observed in samples with cations (lane 2, 4, 6, 8). Amount of cleaved substrates increased by 15 min incubation. Figure 4 shows that differentiation between line 2 and 6 (red lines), and between 4 and 8 (green lines) substrates, showing Zn<sub>2</sub><sup>+</sup> enhanced cleaving efficiency.
Where #1-#4 are immediately loaded after mixing, and #5-#8 are loaded after incubation during 15 min at room temperature. Electrophoresis showed bands which migrated faster than original substrates. The faste migrated bands indicate cleaved substrate, and that were observed in samples with cations (lane #2, #4, #6, #8). Amount of cleaved substrates increased by 15 min incubation. Figure 4 shows that differentiation between lane #2 and #6 (red line), and between #4 and #8 (green line) substrates, showing Zn<sub>2</sub><sup>+</sup> enhanced cleaving efficiency.


{|align=center
{|align=center
|[[Image:Electrophoresis leg cut fitc.jpg|400px|left|thumb|Figure 3. legs and substrate with attached fluorescence protein]]
|[[Image:Electrophoresis leg cut fitc.jpg|400px|left|thumb|Figure 3. Legs and substrate with attached fluorescence protein]]
|[[Image:Graph-Znbbufer.JPG|400px|right|thumb|Figure 4. Plots of differences of luminosity between 0 min and 15min incubated samples under Mg<sub>2</sub><sup>+</sup> (red lines) or Mg<sub>2</sub><sup>+</sup> / Zn<sub>2</sub><sup>+</sup> (green lines) conditions]]
|[[Image:Graph-Znbbufer.JPG|400px|right|thumb|Figure 4. Plots of differences of luminosity between 0 min and 15min incubated samples under Mg<sub>2</sub><sup>+</sup> (red lines) or Mg<sub>2</sub><sup>+</sup> / Zn<sub>2</sub><sup>+</sup> (green lines) conditions]]
|}
|}
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== Method for cutting M13 ==
== Method for cutting M13 ==
[[Image:m13cut　for wiki.jpg|250px|right|thumb|Figure 6. Cutting M13. whole M13 (lane 1).
[[Image:M13cutforwiki.jpg.png|250px|right|thumb|Figure 6. Cut M13. whole M13 (line 1).
M13 after treatment with restriction enzymes (lane 2)]]
M13 after treatment with restriction enzymes (line 2)]]
 
Experiment conditions:


*Gel: 1.0% agarose gel
*Gel: 1.0% agarose gel
*Buffer: 1×TAE
*Buffer: 1×TAE
For producing the robot body, firstly we used whole M13mp18 DNA single strand (M13) as scaffold. However, 1,108 bases of 7,249 bases were used in our design. Thus, no used region of M13 was cut with restriction enzyme (Bal I and PstI). In figure 6, degrees of migration of whole M13 and cut M13 after treatment with restriction enzymes were compared by electrophoresis. 1; whole M13, and 2; M13 after cut treatment. A; the band of whole M13. B; the band of longer cut M13 treatment with restriction enzymes. C; the band of shorter M13 which we use in our DNA origami. D; the band of the short DNA (20base) which was used to form partial double helix to cut M13 by restriction enzymes. The reason why the band of C is smaller amount than the band of D is that the samples was purified by QIAGEN PCR purification kit, by which longer DNA (>5000bp) is not effectively purified, before loading gels. Detailed procedures are described in [http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Notes#M13.E3.81.AE.E5.88.87.E6.96.AD.E3.80.80Cutting_of_M13 '''Here'''].
 
For producing the robot body, we firstly used whole M13mp18 DNA single strand (M13) as scaffold.  
However, 1,108 of 7,249 nucleotides were used in our design (just 16% of the total M13 strand). Thus, the region no necessary to be used was cut with restriction enzyme (Bal I and Pst I). In figure 6, different degrees of migration of the whole M13 and cut M13, after treatment with restriction enzymes, were compared by electrophoresis. Label 1: shows the whole M13, and label 2: the M13 after the cutting treatment. Band A: the band corresponding with the whole M13. Band B: the band corresponding to the longer cut M13 treatment with restriction enzymes. Band C: the band corresponding to the shorter M13 which we use in our DNA origami. Band D: the band of the short DNA (20base) which was used to form partial double helix to cut M13 by restriction enzymes. The reason why the band C is slower than D is because the samples were purified by the QIAGEN PCR purification kit, by which long DNA strands (more than 5000base) are not effectively purified, before loading gels. Detailed procedures are described in [http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Notes#Method_for_cutting_the_M13 '''Here'''].
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About the methods

In this section we are presenting the procedures we took for the following cases:

  • Verifying 2D and 3D structures. Basically we verified whether the folding was properly done or not.
  • Verifying whether the DNAzyme legs cut the substrates. Important step for the robot motion.
  • After the folding, there were some leftover staples. We needed to get rid of them before the next step.
  • Because we cut M13 it is needed to check whether we got the desire 1,108 nucleotides.

Take into consideration that DNA strands were stained by SYBR Gold (sigma) except when observing them with fluorescence.


<html> <center> <TABLE border="0"> <tr> <td> <br/> <table width="90%" border="0"><tr><td style="text-align:center;"> <A Href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis#Method_for_verifying_the_structure"> <Img Src="http://openwetware.org/images/c/cc/Structur.png" Border="0" Width="400" Height="150"><br>Method for verifying the structure</a> </td> <td style="text-align:center;"> <A Href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis#Method_for_verifying_that_the_DNAzyme_legs_cut_the_substrates"> <Img Src="http://openwetware.org/images/1/1c/Leg-cut.png" Border="0" Width="400" Height="150"　align=""right""><br>Method for verifying that the DNAzyme legs cut substrates</a> </td> <td style="text-align:center;"> </table> </td> </tr> </TABLE> </html> <html> <TABLE border="0"> <tr> <td> <br/> <table width="90%" border="0"><tr> <td style="text-align:center;"> <A Href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis#Method_for_removing_remained_staples_after_DNA_origami_folding"> <Img Src="http://openwetware.org/images/f/f4/Remove.png" Border="0" Width="400" Height="150"><br>Method for removing remained staples after DNA origami folding</a> </td> <td style="text-align:center;"> <A Href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis#Method_for_cutting_M13"> <Img Src="http://openwetware.org/images/e/e3/M13_cut.gif" Border="0" Width="240" Height="180"><br>Method for cutting M13</a> </td> <td style="text-align:center;"> </table> </td> </tr> </TABLE> </center> </html>

Method for verifying the structure

Verifying 2D and 3D structure (using whole M13 for the robot body)

Experiment conditions:

  • Gel: 1.4% agarose gel
  • Buffer: 1×TAE Mg2+

We tested the influence of time and ratio to make our 2D and 3D nanostructures, by changing annealing time and concentration ratio between M13 and staples. This staples are the needed for forming 2D and for folding the 2D structure into the 3D structure. From now on, the last kind of staple will be refer as 'staples for forming the 3D structure'. As a result of the experiment, we got that the 2h annealing was better than 3h annealing, and a high concentration of staples (1:100) was better.

Figure 1. 2 hours annealing. Samples indicates, #1: only M13. #2 and #4: M13 with the staples for forming 2D structure (not including staples for forming 3D structure). #3 and #5: M13 and staples for forming 2D structures and 3D structures. #2 and #3: M13:staples (1:10). #4 and #5: M13:staples (1:100).
Figure 2. 3 hours annealing. #1: only M13. 2: M13 with the staples for forming 2D structure (not including staples for forming 3D structure. #3: M13 and staples for forming 2D structures and 3D structures. #2 and #3: M13:staples (1:100)


Verifying the 2D and 3D structure (using cut M13)

Figure 3. Band gaps between 2D and 3D structures. Samples indicate, #1: 100bp DNA ladder marker. #2: cut M13 (234 fmol). #3: 2D structure (development view) not including staples for forming 3D structure. #4: 2D structure including staples for forming the 3D structure.

We carried out electrophoresis to 2D and 3D structure to figure out how the band gap differences among these two are. Figure 3 shows the band gap of 2D and 3D structure.

To examine whether we succeed to construct the designed 2D DNA origami, electrophoresis were performed. Folded 2D and 3D structure were observed as migrations in agarose gels. Figure 3 shows different migrations of 2D and 3D structure by the electrophoresis.

Experiment conditions:

Cut M13: 58.8 nM for each 3.4μL
Staples: 400 nM for each 5μL
5×TAE-Mg2+: 10μL
Distilled water: 31.6μL
Total: 50μL
Annealing: from 90ºC to 25 ºC (decreasing 1 ºC each 3 minutes)

As a result of the electrophoresis: Band C indicates the cut M13. Band B indicates the 2D structure, because this band shows an slower migration than cut M13. Two bands were observed in lane #4, and the lower band showed similar migration as the band in lane #3. There are two possibilities to consider in case of the upper band. First, one of the two bands indicates the band of the triangular prism structure. Second, this is the band which was made by connection of 2D structures between each other. However, if the upper band indicated connection of two pieces of 2D structures, two or more bands should have been observed. Thus, we arrived to the conclusion that the upper band indicates the designed 3D structure. Our AFM observation supports this conclusion. Please see this page for the AFM observation.

Method for verifying that the DNAzyme legs cut the substrates

DNAzyme Legs and a substrate with internal ribonucleotide and fluorescence protein at 5’ end

Experiment conditions:

  • Gel: 24% Poly-Acrylamide Gel including 6M Urea
  • Buffer: 1×TAE
  • Sample: mixtures of DNAzyme Legs and the substrates

In figure 3, samples loaded in each well are: 1 and 5 contain 1xTA
2 and 6 contain 1xTA Mg2+ (12.5mM)
3 and 7 contain 1xTA Zn2+ (1mM)
4 and 8 contain 1xTA Mg2+ (12.5mM) and Zn2+ (1mM)
Where #1-#4 are immediately loaded after mixing, and #5-#8 are loaded after incubation during 15 min at room temperature. Electrophoresis showed bands which migrated faster than original substrates. The faste migrated bands indicate cleaved substrate, and that were observed in samples with cations (lane #2, #4, #6, #8). Amount of cleaved substrates increased by 15 min incubation. Figure 4 shows that differentiation between lane #2 and #6 (red line), and between #4 and #8 (green line) substrates, showing Zn2+ enhanced cleaving efficiency.

Figure 3. Legs and substrate with attached fluorescence protein
Figure 4. Plots of differences of luminosity between 0 min and 15min incubated samples under Mg2+ (red lines) or Mg2+ / Zn2+ (green lines) conditions


Cleaving of substrates without fluorescence by DNAzyme legs

Figure 5. Cleaving of substrates without fluorescence by DNAzyme legs

Experiment conditions:

  • Gel: 24% Poly-Acrylamide Gel with 6 M urea
  • Buffer: 1×TAE
  • Sample: Legs and substrates are loaded on line 1, 2, 3 and 4. Substrates and DNAzyme legs were loaded on 5 and 6, respectively.

Mg2+ (12.5 mM) Zn2+ (1mM), Zn2+ (1mM), and Mg2+ (12.5 mM) were added to the sample of lane in 1, 2 and 3, respectively. After 15 min incubation, samples were loaded on agarose gels. Figure 5 shows that substrates of 1, 2 and 3 are properly cut, indicating that the double helix was formed.

Method for removing remained staples after DNA origami folding

Gel filtration by a micro spin column S-400HR

Figure 5. Gel filtration by S-400HR micro spin column. #1: 100bp DNA ladder marker, #2: 3D structure sample, #3: 3D structure sample through gel filtration

Experiment conditions:

  • Gel: 0.7% agarose gel
  • Buffer: 1×TAE Mg2+

To remove remained staples after the DNA origami fold, a gel filtration was performed using S-H400R micro spin column (GE health care). Through gel filtration, remained staples were totally removed (see figure 5). However, folded structures also diminished. Since folded structure was not observed by AFM, we concluded that the folded structures disrupted through SH400R gel filtration.

polyethylene glycol (PEG) precipitation

Figure 4. PEG precipitation

We carried out a PEG precipitation to get rid of the leftover staples from the sample.

Experiment conditions:

  • 10mM MgCl2, PEG6000 10%, to a sample.
  • At 16,000×g in a conventional tabletop microcentrifuge for 10 minute at RT.
  • Removed Supernatant liquid and a sample is dissolved by 1×TAE-Mg2+ of 80% of a total amount.

It seemed that more staples were removed by two consecutive PEG precipitation.

Method for cutting M13

Figure 6. Cut M13. whole M13 (line 1). M13 after treatment with restriction enzymes (line 2)

Experiment conditions:

  • Gel: 1.0% agarose gel
  • Buffer: 1×TAE

For producing the robot body, we firstly used whole M13mp18 DNA single strand (M13) as scaffold. However, 1,108 of 7,249 nucleotides were used in our design (just 16% of the total M13 strand). Thus, the region no necessary to be used was cut with restriction enzyme (Bal I and Pst I). In figure 6, different degrees of migration of the whole M13 and cut M13, after treatment with restriction enzymes, were compared by electrophoresis. Label 1: shows the whole M13, and label 2: the M13 after the cutting treatment. Band A: the band corresponding with the whole M13. Band B: the band corresponding to the longer cut M13 treatment with restriction enzymes. Band C: the band corresponding to the shorter M13 which we use in our DNA origami. Band D: the band of the short DNA (20base) which was used to form partial double helix to cut M13 by restriction enzymes. The reason why the band C is slower than D is because the samples were purified by the QIAGEN PCR purification kit, by which long DNA strands (more than 5000base) are not effectively purified, before loading gels. Detailed procedures are described in Here.

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