Difference between revisions of "Team:Vilnius-Lithuania/Protocols"

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<h4 class="page-heading">Agarose Gel preparation (x%)</h4>
 
<h4 class="page-heading">Agarose Gel preparation (x%)</h4>
 
<h4 class="page-heading">Agarose Gel preparation (x%)</h4>
 
<ol>
 
<ol>
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<li>An expression system to maximize secreted toxic protein yields</li><br>
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<li>To prepare agarose gels we used TopVision Agarose Tablets that are manufactured by Thermo Fisher Scientific.</li>
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<li>A reporter-based system for observing the protein of interest dynamics (expression level, degradation, aggregation, and toxicity effect on a cell) in real-time.</li><br>
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<li>Add appropriate number of agarose tablets to the electrophoresis buffer based on the table below to prepare your desired gel percentage</li>
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<li>Biological polymers as a way of information storage</li><br>
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<li>Adaptive laboratory evolution for optimization of strains synthesizing membrane proteins</li><br>
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<p>
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<li>Repressor-based light-inducible one-component systems </li><br>
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Note: Use a flask that is 2 to 4 times the volume of the solution being prepared.</p>
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<li><i>De novo</i> enzymatic DNA synthesis</li><br>
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<li><i>In silico</i> metagenomic mining</li><br>
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<li>Before heating soak tablets in a buffer (~ 4 minutes) until tablets completely break into fine-particle slurry. Swirl the slurry to break up any remaining particles. Important: Ensure tablets break up entirely. Heating will render non-dispersed agarose particles insoluble.</li>
 +
Note: Heating times are dependent on the volume of liquid and number of gel tablets to dissolve.
 +
<li>Remove the flask from microwave, swirl gently to dissolve any remaining agarose particles.</li>
 +
<li>Reheat on high power for 1-2 minutes or until the solution is clear and all particles are dissolved.</li>
 +
<li>Remove the flask from the microwave oven, and gently swirl.</li>
 +
<li>Cool the solution to approximately 50-60 °C.</li>
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<li>Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). Mix well.</li>
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<li>Pour the gel into a tray of required size and place in the well comb, let the gel cool and solidify for 10-15 mins at room temperature.</li>
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<li>This gel can now be used to run electrophoresis gels.</li>
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<p>
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Note: To let the gel cool down to the required temperature of about 60 °C one can use a smaller volume container, which is not affected by heat to let a smaller volume of the melted agarose cooldown faster.</p>
 
</ol>
 
</ol>
 
<br>
 
<br>

Revision as of 23:18, 20 October 2019

Protocols

Agarose Gel preparation (x%)

Agarose Gel preparation (x%)

  1. To prepare agarose gels we used TopVision Agarose Tablets that are manufactured by Thermo Fisher Scientific.
  2. Add appropriate number of agarose tablets to the electrophoresis buffer based on the table below to prepare your desired gel percentage

    3. Note: Use a flask that is 2 to 4 times the volume of the solution being prepared.

  3. Before heating soak tablets in a buffer (~ 4 minutes) until tablets completely break into fine-particle slurry. Swirl the slurry to break up any remaining particles. Important: Ensure tablets break up entirely. Heating will render non-dispersed agarose particles insoluble.
    4. Note: Heating times are dependent on the volume of liquid and number of gel tablets to dissolve.
  4. Remove the flask from microwave, swirl gently to dissolve any remaining agarose particles.
  5. Reheat on high power for 1-2 minutes or until the solution is clear and all particles are dissolved.
  6. Remove the flask from the microwave oven, and gently swirl.
  7. Cool the solution to approximately 50-60 °C.
  8. Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). Mix well.
  9. Pour the gel into a tray of required size and place in the well comb, let the gel cool and solidify for 10-15 mins at room temperature.
  10. This gel can now be used to run electrophoresis gels.

    11. Note: To let the gel cool down to the required temperature of about 60 °C one can use a smaller volume container, which is not affected by heat to let a smaller volume of the melted agarose cooldown faster.


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