Difference between revisions of "Team:Freiburg/Protocols/Agarose-Gel"

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<h1 class="sectionedit1">Agarose gels</h1>
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<h1 class="sectionedit1">Agarose Gel Electrophoresis</h1>
 
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<strong> Protocol for casting and loading of a DNA fractionation gel</strong>
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Agarose Gel Electrophoresis is a commonly used method to analyze the length of DNA fragments. It is suitable to confirm succesful amplification of DNA fragments by PCR as well as analysis of restriction digests. DNA fragments can be extracted from the gel afterwards (see: Gel Extraction protocol). <br>
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The phosphate backbone applies a negative charge to the DNA. Therefore, an electric field can be used to move DNA. The polymerization of agarose results in a close net which hinders the DNA from being moved by the electric force. Longer DNA fragments migrate even slower than shorter fragments. Thus, the distance a DNA fragment moves in a certain time is correlated to its length. Using a ladder containing fragments of defined lengths enables to estimate the absolute length of the fragment of interest.
 
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<img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png"/> material: TAE-Puffer (0.5x), Agarose, microwave<br/>
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<img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png"/> Material: TAE-Puffer (1x), agarose, microwave<br/>
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<img alt="" class="media" src="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png"/> duration: 90 min<br/>
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<img alt="" class="media" src="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png"/> Duration: 90 min (30 min for agarose preparation, 30 min for polymerization and 30 min for actual sample analysis)<br/>
 
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<strong> Casting </strong>
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<strong> Preparation </strong>
 
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<ol>
 
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<li class="level1"><div class="li"> pour 0.7% or 1% agarose solution into the casting chamber depending on the size of the DNA fragments (the higher concentration, the smaller the fragment)</div>
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<li>The appropriate concentration of agarose depends on the size of the fragments to be analyzed (the smaller the fragments, the higher the agarose concentration).</li>
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</li>
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<li>Weigh out the appropriate amount of agarose add it to 1x TAE buffer (for example: 1 g agarose in 100 ml TAE results in a 1% agarose gel, which we commonly used for intermediate length fragments)</li>
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<li class="level1"><div class="li"> add <strong>4 µL</strong> of GelRed solution and distribute it by using the gel comb</div>
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<li>To solve the agarose, heat it in a microwave until the solution is completely clear (<strong>Be aware of boiloing retardation!</strong>)</li>
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</li>
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<li>Pour agarose solution into the casting chamber.</li>
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<li class="level1"><div class="li"> fix comb in the gel chamber</div>
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<li>Add GelRed for a final concentration of 1x (10.000x stock solution) and distribute it by using the gel comb (<strong>Caution: GelRed is a mutagenic!</strong>).</li>
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</li>
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<li>Fix comb in the gel chamber.</li>
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<li class="level1"><div class="li"> <strong>let polymerize</strong></div>
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<li>Let the agarose cool down and polymerize.</li>
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</li>
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<li>Fill gel chamber with 1x TAE-Buffer.</li>
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<li class="level1"><div class="li"> Fill gel chamber with TAE-Buffer</div>
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<li>Carefully remove the comb.</li>
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</li>
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<li class="level1"><div class="li"> <strong>remove comb</strong> (beware for cracks)</div>
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CAUTION: hot! ⇒ use gloves for handling<br/>
 
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<strong>CAUTION: GelRed ist ein strong mutagen!!</strong>
 
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<strong> Sample analysis </strong>
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<strong>Gel loading </strong>
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<li class="level1"><div class="li"> <strong>add 4 µL</strong> of ladder into one to two gel pockets (depending on the size of the gel)</div>
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<li>Add 4 µL DNA ladder into one to two gel pockets (depending on the size of the gel).</li>
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</li>
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<li>Add loading dye to your DNA samples for final concentration of 1x.</li>
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<li class="level1"><div class="li"> add <strong>running buffer</strong> to the DNA samples (6x concentrated)</div>
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<li>Pipette DNA samples in the gel pockets (volume depending on the size of the gel) and avoid air bubbles.</li>
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</li>
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<li>Close the lid and apply voltage to the chamber (dependent on the size of the fragments and the resolution needed; commonly 100 to 120 V)</li>
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<li class="level1"><div class="li"> load <strong>DNA samples</strong> in the gel pockets (volume depending on the size of the gel)</div>
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<li> Run the gel until the loading dye reaches the lower end of the gel.</li>
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</li>
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<li class="level1"><div class="li"> <strong>apply voltage</strong> → 120V</div>
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</li>
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<li class="level1"><div class="li"> Running time depends on the desired quality of band splitting</div>
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<strong> Cast a new gel </strong>
 
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<li class="level1"><div class="li"> <strong>weight </strong> agarose (depending on concentration) and add the corresponding amount of water</div>
 
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</li>
 
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<li class="level1"><div class="li"> <strong>heat</strong> in the microwave until the solution is has cleared up. Pan from time to time to prevent boiling retardation</div>
 
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CAUTION: Beware boiling retardation and NEVER close the cap<br/>
 
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CAUTION: hot! ⇒ use gloves for panning<br/>
 
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Revision as of 16:12, 16 September 2015

""

Agarose Gel Electrophoresis

Agarose Gel Electrophoresis is a commonly used method to analyze the length of DNA fragments. It is suitable to confirm succesful amplification of DNA fragments by PCR as well as analysis of restriction digests. DNA fragments can be extracted from the gel afterwards (see: Gel Extraction protocol).
The phosphate backbone applies a negative charge to the DNA. Therefore, an electric field can be used to move DNA. The polymerization of agarose results in a close net which hinders the DNA from being moved by the electric force. Longer DNA fragments migrate even slower than shorter fragments. Thus, the distance a DNA fragment moves in a certain time is correlated to its length. Using a ladder containing fragments of defined lengths enables to estimate the absolute length of the fragment of interest.

Material: TAE-Puffer (1x), agarose, microwave
Duration: 90 min (30 min for agarose preparation, 30 min for polymerization and 30 min for actual sample analysis)

Preparation

  1. The appropriate concentration of agarose depends on the size of the fragments to be analyzed (the smaller the fragments, the higher the agarose concentration).
  2. Weigh out the appropriate amount of agarose add it to 1x TAE buffer (for example: 1 g agarose in 100 ml TAE results in a 1% agarose gel, which we commonly used for intermediate length fragments)
  3. To solve the agarose, heat it in a microwave until the solution is completely clear (Be aware of boiloing retardation!)
  4. Pour agarose solution into the casting chamber.
  5. Add GelRed for a final concentration of 1x (10.000x stock solution) and distribute it by using the gel comb (Caution: GelRed is a mutagenic!).
  6. Fix comb in the gel chamber.
  7. Let the agarose cool down and polymerize.
  8. Fill gel chamber with 1x TAE-Buffer.
  9. Carefully remove the comb.



Sample analysis

  1. Add 4 µL DNA ladder into one to two gel pockets (depending on the size of the gel).
  2. Add loading dye to your DNA samples for final concentration of 1x.
  3. Pipette DNA samples in the gel pockets (volume depending on the size of the gel) and avoid air bubbles.
  4. Close the lid and apply voltage to the chamber (dependent on the size of the fragments and the resolution needed; commonly 100 to 120 V)
  5. Run the gel until the loading dye reaches the lower end of the gel.