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>
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<ul>
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<ol>
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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>An expression system to maximize secreted toxic protein yields</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>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>Biological polymers as a way of information storage</li><br>
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<p>
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<li>Adaptive laboratory evolution for optimization of strains synthesizing membrane proteins</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>Repressor-based light-inducible one-component systems </li><br>
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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>
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</ol>
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Note: Heating times are dependent on the volume of liquid and number of gel tablets to dissolve.
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<li>Remove the flask from microwave, swirl gently to dissolve any remaining agarose particles.</li>
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<li>Reheat on high power for 1-2 minutes or until the solution is clear and all particles are dissolved.</li>
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<li>Remove the flask from the microwave oven, and gently swirl.</li>
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<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>
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</ul>
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Revision as of 23:11, 20 October 2019

Protocols

Agarose Gel preparation (x%)

  1. An expression system to maximize secreted toxic protein yields

  2. A reporter-based system for observing the protein of interest dynamics (expression level, degradation, aggregation, and toxicity effect on a cell) in real-time.

  3. Biological polymers as a way of information storage

  4. Adaptive laboratory evolution for optimization of strains synthesizing membrane proteins

  5. Repressor-based light-inducible one-component systems

  6. De novo enzymatic DNA synthesis

  7. In silico metagenomic mining


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