Difference between revisions of "Team:TU Dresden/Notebook/Protocols/AgaroseGel"

 
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<div id="TueContent">
  
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<h2>How to prepare a 0.7 % agarose gel</h2>
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<h2>Preparation of 0.7% and 2% agarose gel</h2>
  
 
<p style="line-height:1.8;><b>Wear nitrile gloves when preparing the gel, ethidium bromide is considered toxic. </b></p>
 
<p style="line-height:1.8;><b>Wear nitrile gloves when preparing the gel, ethidium bromide is considered toxic. </b></p>
  
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Recipe for 10xTBE buffer:
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<p style="line-height:1.8;>Recipe for 10x TBE buffer:</p>
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<ul>
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0.89 M Boric acid,      Mr 121.14
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  <li style="margin-bottom: 10px;line-height:1.8;">0.89 M Boric acid (M<sub>r</sub> 121.14).</li>
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0.89 M Tris,                Mr  61.83
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  <li style="margin-bottom: 10px;line-height:1.8;">0.89 M Tris (M<sub>r</sub> 61.83).</li>
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0.02 M EDTA pH 8.0   500 mM stock solution pH 8.0  
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  <li style="margin-bottom: 10px;line-height:1.8;">0.02 M EDTA pH 8.0 500mM stock solution pH 8.0.</li>
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</ul>
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<ol>
 
<ol>
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   <li style="margin-bottom: 10px;line-height:1.8;"></li>
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   <li style="margin-bottom: 10px;line-height:1.8;">Dissolve 0.42 g of agarose (0.7%) or 1.2 g of agarose (2%) in 60 mL of 1 x TBE buffer.</li>
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   <li style="margin-bottom: 10px;line-height:1.8;"></li>
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   <li style="margin-bottom: 10px;line-height:1.8;">Use TBE for making analytical gels and use TAE buffer if gel is meant for DNA or plasmid extraction.</li>
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   <li style="margin-bottom: 10px;line-height:1.8;">&mu;L &deg;C </li>
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   <li style="margin-bottom: 10px;line-height:1.8;">Melt the agarose in the microwave until boiling.</li>
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   <li style="margin-bottom: 10px;line-height:1.8;"></li>
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  <li style="margin-bottom: 10px;line-height:1.8;">Take out the solution and mix it by shaking the bottle.</li>  
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   <li style="margin-bottom: 10px;line-height:1.8;"></li>
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   <li style="margin-bottom: 10px;line-height:1.8;">Put the solution back into the microwave and let it boil again.</li>
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   <li style="margin-bottom: 10px;line-height:1.8;"></li>
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   <li style="margin-bottom: 10px;line-height:1.8;">Repeat mixing and boiling until the agarose is completely melted.</li>
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   <li style="margin-bottom: 10px;line-height:1.8;"></li>
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   <li style="margin-bottom: 10px;line-height:1.8;">Set up an agarose gel box with spacers and a comb.</li>
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   <li style="margin-bottom: 10px;line-height:1.8;"></li>
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   <li style="margin-bottom: 10px;line-height:1.8;">Take out some solution with a blue pipette tip and put it along the inside of the spacers in the gel box and let it solidify.</li>
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   <li style="margin-bottom: 10px;line-height:1.8;"></li>
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   <li style="margin-bottom: 10px;line-height:1.8;">Add 2 &mu;L of Ethidium Bromide to the remaining solution and mix by shaking the bottle.</li>
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   <li style="margin-bottom: 10px;line-height:1.8;"></li>
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   <li style="margin-bottom: 10px;line-height:1.8;">Pour the rest of the solution into the gel box.</li>
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   <li style="margin-bottom: 10px;line-height:1.8;">Let it stand for at least 30 minutes until the gel becomes solid.</li>
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  <li style="margin-bottom: 10px;line-height:1.8;">Fill the gel box up with 1x TBE (or 1x TAE) buffer so that the gel is just covered.</li>
 
</ol>
 
</ol>
  

Latest revision as of 23:24, 13 September 2015


Preparation of 0.7% and 2% agarose gel

Recipe for 10x TBE buffer:

  • 0.89 M Boric acid (Mr 121.14).
  • 0.89 M Tris (Mr 61.83).
  • 0.02 M EDTA pH 8.0 500mM stock solution pH 8.0.
  1. Dissolve 0.42 g of agarose (0.7%) or 1.2 g of agarose (2%) in 60 mL of 1 x TBE buffer.
  2. Use TBE for making analytical gels and use TAE buffer if gel is meant for DNA or plasmid extraction.
  3. Melt the agarose in the microwave until boiling.
  4. Take out the solution and mix it by shaking the bottle.
  5. Put the solution back into the microwave and let it boil again.
  6. Repeat mixing and boiling until the agarose is completely melted.
  7. Set up an agarose gel box with spacers and a comb.
  8. Take out some solution with a blue pipette tip and put it along the inside of the spacers in the gel box and let it solidify.
  9. Add 2 μL of Ethidium Bromide to the remaining solution and mix by shaking the bottle.
  10. Pour the rest of the solution into the gel box.
  11. Let it stand for at least 30 minutes until the gel becomes solid.
  12. Fill the gel box up with 1x TBE (or 1x TAE) buffer so that the gel is just covered.