Difference between revisions of "Team:Grenoble-Alpes/purification module"

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<p>In our automated system, a crucial step is to extract DNA in order to get through our detection process. Indeed at the end of the lysis step, our solution contains a lot of cellular debris that could prevent the process from being carried out correctly.</p><p>
 
<p>In our automated system, a crucial step is to extract DNA in order to get through our detection process. Indeed at the end of the lysis step, our solution contains a lot of cellular debris that could prevent the process from being carried out correctly.</p><p>
 
A lot of different methods can be used to purify DNA in a lab. The most common example is the use of a silica minicolumn DNA purification. </p>
 
A lot of different methods can be used to purify DNA in a lab. The most common example is the use of a silica minicolumn DNA purification. </p>
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Looking at all those purifications methods from an engineering point of view, the main common issue is the need for a centrifugation step at some point in the process. As it is difficult to obtain the necessary force to extract DNA in a small low-cost automated system, we looked for an alternative way and that’s how we discovered about magnetic beads. </p>
+
<p>Looking at all those purifications methods from an engineering point of view, the main common issue is the need for a centrifugation step at some point in the process. As it is difficult to obtain the necessary force to extract DNA in a small low-cost automated system, we looked for an alternative way and that’s how we discovered about magnetic beads. </p>
  
 
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Click on the button below to get more details about how magnetic beads work, and the different washing steps of a DNA extraction using them.
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<p>Click on the button below to get more details about how magnetic beads work, and the different washing steps of a DNA extraction using them. </p>
  
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<br>
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<div class="collapse slide">
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<h2><font size="5">About the magbeads</font></h2>
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<p><b>The coating :</b></p>
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The main goal of the coating is to bind with DNA. The most commonly used coating are SiO2 and TiO2.
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In the case of SiO2, the binding with DNA can be control through the concentration of salt (ions) in solution : in a high salt solution the SiO2 coating is positively charged and will therefore attach to DNA thanks to Van der Waals interactions ; in a low salt solution SiO2 will repel DNA because of electrostatic forces.
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<br>
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<figure><center><img src="https://static.igem.org/mediawiki/2018/f/f7/T--Grenoble-Alpes--fluofig3.png"><figcaption>Figure 2: Schematics of a magnetic bead  </figcaption></center></figure>
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<br>
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Magnetic core & Field :
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Once the DNA is attached to the beads, we want to move those around, this is done by applying a magnetic field (in our case through permanent magnet). The beads will move from the region with a low magnetic field to the region with a high magnetic field.
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<br>
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<figure><center><img src="https://static.igem.org/mediawiki/2018/f/f7/T--Grenoble-Alpes--fluofig3.png"><figcaption>Figure 2: Schematics of a magnetic bead  </figcaption></center></figure>
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<br>
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The different steps of magnetic beads DNA purification:
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1) Magnetic beads are put into the solution containing a lot of residues and the DNA that we want to extract. We add a high salt solution to enable the binding between the beads and DNA.
 +
 +
<br>
 +
<figure><center><img src="https://static.igem.org/mediawiki/2018/f/f7/T--Grenoble-Alpes--fluofig3.png"><figcaption>Figure 2: Schematics of a magnetic bead  </figcaption></center></figure>
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<br>
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2)  DNA binds to the beads.
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<br>
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<figure><center><img src="https://static.igem.org/mediawiki/2018/f/f7/T--Grenoble-Alpes--fluofig3.png"><figcaption>Figure 2: Schematics of a magnetic bead  </figcaption></center></figure>
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<br>
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3) a magnetic field is applied to move the beads to the edge.
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<br>
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<figure><center><img src="https://static.igem.org/mediawiki/2018/f/f7/T--Grenoble-Alpes--fluofig3.png"><figcaption>Figure 2: Schematics of a magnetic bead  </figcaption></center></figure>
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<br>
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4) we wash beads removing solution and adding  new high salt solution by automatic pipetting. (DNA is still located on the edge through those washing steps)
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For those washes we continue to use a high salt solution to keep DNA bound to the beads.
 +
 +
<br>
 +
<figure><center><img src="https://static.igem.org/mediawiki/2018/f/f7/T--Grenoble-Alpes--fluofig3.png"><figcaption>Figure 2: Schematics of a magnetic bead  </figcaption></center></figure>
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<br>
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5) The final wash is done with a low salt solution : the DNA begin to detach itself from the beads. We remove the magnetic field in order to release  all the DNA into the solution.
 +
<br>
 +
<figure><center><img src="https://static.igem.org/mediawiki/2018/f/f7/T--Grenoble-Alpes--fluofig3.png"><figcaption>Figure 2: Schematics of a magnetic bead  </figcaption></center></figure>
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<br>
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  6) We apply once again the magnetic field (still with a low salt solution), and we pipette  the center of the tube : this way we recover the DNA but not the beads.
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Issues that one can encounter using magnetic beads purification :
 +
 +
Sedimentation: beads that are too large will fall to the bottom of the tube, they will no longer be retrievable, this problem is avoided by limiting the time during which the balls are pulled.
 +
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Pipetting: Good pipetting accuracy is required to avoid inadvertently pipetting beads. A biologist should be careful not to shake when pipetting, a machine should be sufficiently stable and placed well in the center of the hole.
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</div>
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<div class="collapse slide">
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<h2><font size="5">About the magnetic field</font></h2>
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In order to create a magnetic field we can either use permanent magnets, or electromagnets.
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Permanent magnets can be really strong, but they need to be physically moved in order to apply or not a magnetic field.
 +
On the other hand electromagnets are weaker but can be turned on and off through a numerical controller.
 +
 +
In order to reduce the amount of troubleshooting we would prefer electromagnets, as no mechanical action is required. However the electromagnets that we tried to use or create were too weak to catch the magnetic beads. We therefore had to use permanent magnets.
 +
 +
 +
To move the magnets we use an actuator, which is a motor allowing linear movement. The motor is placed directly on the rotative plate, in order to be sure of the relative position of the magnets compared to the purification tubes. We also added a support system of the piece motor to be sure it slides correctly to the tubes.
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To place the magnets as close as possible from the tube we also tailored the lysis aluminum block in this particular way :
  
  
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Revision as of 14:33, 14 October 2018

Template loop detected: Template:Grenoble-Alpes

PURIFICATION MODULE

In our automated system, a crucial step is to extract DNA in order to get through our detection process. Indeed at the end of the lysis step, our solution contains a lot of cellular debris that could prevent the process from being carried out correctly.

A lot of different methods can be used to purify DNA in a lab. The most common example is the use of a silica minicolumn DNA purification.

Looking at all those purifications methods from an engineering point of view, the main common issue is the need for a centrifugation step at some point in the process. As it is difficult to obtain the necessary force to extract DNA in a small low-cost automated system, we looked for an alternative way and that’s how we discovered about magnetic beads.


Figure 1: Schematics of a magnetic bead

Magnetic beads are very small (1 µm) particles, consisting of a ferromagnetic core and a coating.

The coating of the beads is used to attach the DNA in the solution, whereas the ferromagnetic core is used to move the beads to the edge of the tube. It allows us to extract DNA by applying a magnetic field following by a few washing steps.

In order to apply the magnetic field, we used small permanent magnets and an actuator. The magnets are crimped in a 3D printed part, which is fixed to the motor. Thanks to this system we can place the magnets closer or further away from the tubes, allowing us with the pipette to automate the DNA extraction.


Figure 2: Schematics of a magnetic bead

Click on the button below to get more details about how magnetic beads work, and the different washing steps of a DNA extraction using them.


About the magbeads

The coating :

The main goal of the coating is to bind with DNA. The most commonly used coating are SiO2 and TiO2. In the case of SiO2, the binding with DNA can be control through the concentration of salt (ions) in solution : in a high salt solution the SiO2 coating is positively charged and will therefore attach to DNA thanks to Van der Waals interactions ; in a low salt solution SiO2 will repel DNA because of electrostatic forces.
Figure 2: Schematics of a magnetic bead

Magnetic core & Field : Once the DNA is attached to the beads, we want to move those around, this is done by applying a magnetic field (in our case through permanent magnet). The beads will move from the region with a low magnetic field to the region with a high magnetic field.
Figure 2: Schematics of a magnetic bead

The different steps of magnetic beads DNA purification: 1) Magnetic beads are put into the solution containing a lot of residues and the DNA that we want to extract. We add a high salt solution to enable the binding between the beads and DNA.
Figure 2: Schematics of a magnetic bead

2) DNA binds to the beads.
Figure 2: Schematics of a magnetic bead

3) a magnetic field is applied to move the beads to the edge.
Figure 2: Schematics of a magnetic bead

4) we wash beads removing solution and adding new high salt solution by automatic pipetting. (DNA is still located on the edge through those washing steps) For those washes we continue to use a high salt solution to keep DNA bound to the beads.
Figure 2: Schematics of a magnetic bead

5) The final wash is done with a low salt solution : the DNA begin to detach itself from the beads. We remove the magnetic field in order to release all the DNA into the solution.
Figure 2: Schematics of a magnetic bead

6) We apply once again the magnetic field (still with a low salt solution), and we pipette the center of the tube : this way we recover the DNA but not the beads. Issues that one can encounter using magnetic beads purification : Sedimentation: beads that are too large will fall to the bottom of the tube, they will no longer be retrievable, this problem is avoided by limiting the time during which the balls are pulled. Pipetting: Good pipetting accuracy is required to avoid inadvertently pipetting beads. A biologist should be careful not to shake when pipetting, a machine should be sufficiently stable and placed well in the center of the hole.

About the magnetic field

In order to create a magnetic field we can either use permanent magnets, or electromagnets. Permanent magnets can be really strong, but they need to be physically moved in order to apply or not a magnetic field. On the other hand electromagnets are weaker but can be turned on and off through a numerical controller. In order to reduce the amount of troubleshooting we would prefer electromagnets, as no mechanical action is required. However the electromagnets that we tried to use or create were too weak to catch the magnetic beads. We therefore had to use permanent magnets. To move the magnets we use an actuator, which is a motor allowing linear movement. The motor is placed directly on the rotative plate, in order to be sure of the relative position of the magnets compared to the purification tubes. We also added a support system of the piece motor to be sure it slides correctly to the tubes. To place the magnets as close as possible from the tube we also tailored the lysis aluminum block in this particular way :