Biomod/2014/Attachement: Difference between revisions

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     <th bgcolor="#be1e3c"><center><font size="+1"><a href="Team"><span style="color:white">Team</span></a></font></center></th>
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   <font size="+1pt">Binding Nanoparticles to the DNA-Origami</font>
   <font size="+1pt">Binding Nanoparticles to the DNA origami</font>
  </p>
  </p>
  <p align="justify" style="line-height:2em"><font size="3pt">To bind the Pt-nanoparticles to the DNA-origami 4 µL of the purified DNA-origami were mixed with 6 µL of the functionalized Pt-nanoparticles and incubated overnight.
  <p align="justify" style="line-height:2em"><font size="3pt">
To bind the Pt-nanoparticles to the DNA origami 4 µL of the purified DNA origami solution were mixed with 6 µL of the functionalized Pt-nanoparticles and incubated overnight.
   <br>
   <br>
At the next day the solution was filtered to remove the excess of Pt-nanoparticles. 10 µL of the DNA origami solution were mixed with 400 µL of the buffer solution (PBS + 100 mM NaCl and 12 mM MgCl<sub>2</sub>) and centrifuged at 5 krcf and 4°C for 5 minutes in a 100K Amicon<sup>®</sup> filter. To recover the DNA-origami from the filter, the filter was turned upside down and centrifuged for 3 min at 1 krcf.</font>
At the next day, the solution was filtered to remove the excess of Pt-nanoparticles. 10 µL of the DNA origami solution were mixed with 400 µL of the buffer solution (PBS + 100 mM NaCl and 12 mM MgCl<sub>2</sub>) and centrifuged at 5 krcf and 4 °C for 5 minutes in a 100K Amicon<sup>®</sup> filter. This was repeated 3 times. Afterwards the filter was flipped to get the bounded DNA origami out of it and centrifuged 3 min at 1 krcf.</font>
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  <p align="justify" style="line-height:1.5em">To proof the attachment of the platinum particles to the Nanoscooter gel electrophoresis is used.
  <p align="justify" style="line-height:1.5em">
   <br> We used a 0.7 % agarose gel and checked for differences between Pt-particles bound to the Nanoscooter and the Pt-particles alone. For the gel, 0.35 g of agarose were added to 50 mL 0.5x TBE buffer and heated for 2 minutes in the microwave oven. After cooling down 2 µL Sybr<sup>®</sup> Safe DNA Gel Stain and 800 µL 16 mM aqueous MgCl<sub>2</sub> solution were added to the agarose gel. Each sample is mixed with 3 µL 10x BlueJuice<sup>TM</sup> Gel Loading buffer.
To verify the attachment of the platinum nanoparticles to the Nanoscooter agarose gel electrophoresis was used.
   <br>We used a 0.7 % agarose gel and checked for differences between Pt-nanoparticles bound to the Nanoscooter and the Pt-nanoparticles alone. For the gel, 0.35 g of agarose were added to 50 mL 0.5x TBE buffer and heated for 2 minutes in the microwave oven. After some cooling down 2 µL Sybr<sup>®</sup> Safe DNA Gel Stain and 800 µL 1 M aqueous MgCl<sub>2</sub> solution were added to the agarose gel. After pouring this mixture into the gel chamber it was waited until the gel got solid.
The samples were prepared like follows:
  </p>
  </p>
   <br>
   <br>
Samples:
Samples:
   <br> 1. 10 µL DNA origami + 10 µL functionalized platinum particles + 3 µL 10X BlueJuice<sup>TM</sup> Gel Loading buffer
   <br> 1. 10 µL DNA origami + 10 µL functionalized platinum nanoparticles (incubated over night) + 3 µL 10X BlueJuice<sup>TM</sup> Gel Loading buffer
   <br>
   <br>
2. 10 µL functionalized platinum particles + 10 µL TE buffer + 11 mM MgCl<sub>2</sub> + 3 µL 10X BlueJuice<sup>TM</sup> Gel              Loading buffer<br>
2. 10 µL functionalized platinum nanoparticles + 10 µL TE buffer with 11 mM MgCl<sub>2</sub> + 3 µL 10X BlueJuice<sup>TM</sup> Gel              Loading buffer<br>
  <p align="justify" style="line-height:1.5em">The gel electrophoresis was carried out for 90 min and a potential of 85 V. After that the gel was analyzed under UV radiation. <font size="3pt">
  <p align="justify" style="line-height:1.5em">The agarose gel electrophoresis was carried out for 90 min and a voltage of 85 V. <font size="3pt">
  </p>
  </p>
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<div align="center"><img src="http://openwetware.org/images/5/5a/Particle-Attachement-Gel.png" width="" height="" ></div>
<div align="center"><img src="http://openwetware.org/images/5/5a/Particle-Attachement-Gel.png" width="" height="" ></div>


<i><font size="3 pt"><div align="center">Figure 1: Gel electrophoresis of the Nanoscooter + platinum particles (slot 1) and the platinum particles (slot 2).</font></div><br></i>
<i><font size="3 pt"><div align="center">Figure 1: Gel electrophoresis of the Nanoscooter + platinum nanoparticles (pocket 1) and the platinum nanoparticles (pocket 2).</font></div><br></i>




<font size="3pt">
<font size="3pt">
  <p align="justify" style="line-height:1.5em">As it can be seen in figure 1 the band of the Nanoscooter with platinum particles (slot 1) runs less far than the platinum particles itself (slot 2). This proves that the attachment of the nanoparticles to the Nanoscooter was successful.</p></font>
  <p align="justify" style="line-height:1.5em">As it can be seen in figure 1 the band of the Nanoscooter with platinum nanoparticles (pocket 1) runs slower than the platinum nanoparticles itself (pocket 2). This proves that the attachment of the nanoparticles to the Nanoscooter was successful.</p></font>
  </p>
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Latest revision as of 14:52, 25 October 2014

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<p align="justify" style="line-height:2em">
  <font size="+2pt"><u>Pt-Particle Attachment</u></font>
 <br>
 <br>
  <font size="+1pt">Binding Nanoparticles to the DNA origami</font>
</p>
<p align="justify" style="line-height:2em"><font size="3pt">

To bind the Pt-nanoparticles to the DNA origami 4 µL of the purified DNA origami solution were mixed with 6 µL of the functionalized Pt-nanoparticles and incubated overnight. 

 <br>

At the next day, the solution was filtered to remove the excess of Pt-nanoparticles. 10 µL of the DNA origami solution were mixed with 400 µL of the buffer solution (PBS + 100 mM NaCl and 12 mM MgCl<sub>2</sub>) and centrifuged at 5 krcf and 4 °C for 5 minutes in a 100K Amicon<sup>®</sup> filter. This was repeated 3 times. Afterwards the filter was flipped to get the bounded DNA origami out of it and centrifuged 3 min at 1 krcf.</font> 

</p>
 <br>
 <br>

   <font size="+1pt">Gel electrophoresis</font>
 <br>
   <font size="3pt">
<p align="justify" style="line-height:1.5em">

To verify the attachment of the platinum nanoparticles to the Nanoscooter agarose gel electrophoresis was used. 

 <br>We used a 0.7 % agarose gel and checked for differences between Pt-nanoparticles bound to the Nanoscooter and the Pt-nanoparticles alone. For the gel, 0.35 g of agarose were added to 50 mL 0.5x TBE buffer and heated for 2 minutes in the microwave oven. After some cooling down 2 µL Sybr<sup>®</sup> Safe DNA Gel Stain and 800 µL 1 M aqueous MgCl<sub>2</sub> solution were added to the agarose gel. After pouring this mixture into the gel chamber it was waited until the gel got solid.

The samples were prepared like follows: 

</p>
 <br>

Samples: 

 <br> 	1. 10 µL DNA origami + 10 µL functionalized platinum nanoparticles (incubated over night) + 3 µL 10X BlueJuice<sup>TM</sup> Gel Loading buffer
 <br>

2. 10 µL functionalized platinum nanoparticles + 10 µL TE buffer with 11 mM MgCl<sub>2</sub> + 3 µL 10X BlueJuice<sup>TM</sup> Gel Loading buffer<br> 

<p align="justify" style="line-height:1.5em">The agarose gel electrophoresis was carried out for 90 min and a voltage of 85 V. <font size="3pt">
</p>
 <br>

<div align="center"><img src="http://openwetware.org/images/5/5a/Particle-Attachement-Gel.png" width="" height="" ></div>

<i><font size="3 pt"><div align="center">Figure 1: Gel electrophoresis of the Nanoscooter + platinum nanoparticles (pocket 1) and the platinum nanoparticles (pocket 2).</font></div><br></i>


<font size="3pt"> 

<p align="justify" style="line-height:1.5em">As it can be seen in figure 1 the band of the Nanoscooter with platinum nanoparticles (pocket 1) runs slower than the platinum nanoparticles itself (pocket 2). This proves that the attachment of the nanoparticles to the Nanoscooter was successful.</p></font>
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