Difference between revisions of "Team:Paris Bettencourt/Protocols/Gel Electrophoresis"

 
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         <ul>
 
         <ul>
 
           <li>Mix 0.5 grams of agarose with 50mL 0.5% TAE solution</li>
 
           <li>Mix 0.5 grams of agarose with 50mL 0.5% TAE solution</li>
−
           <li>Heat the Erlenmeyer in a microwave until the liquid becomes totally transparent.</li>
+
           <li>Heat the Erlenmeyer (conical flask) in a microwave until the liquid becomes totally transparent.</li>
 
           <li>Let it rest until you can take the Erlenmeyer in your hand without pain for 10 seconds.</li>
 
           <li>Let it rest until you can take the Erlenmeyer in your hand without pain for 10 seconds.</li>
−
           <li>Add 5uL of SYBR® Safe (10000X) and mix gently until the liquid takes and uniform reddish color.</li>
+
           <li>Add 5uL of SYBR® Safe (10000X) and mix gently until the solution homogenizes.</li>
−
           <li>Pour the liquid in a gel mold with the desired number of well and wait 20-30 min (or until the gel is solidified).</li>
+
           <li>Pour the liquid in a gel mold with the desired number of wells and wait 20-30 min (or until the gel is solidified).</li>
−
           <li>Put the gel inside the electrophoresis machine and pour some TAE 0.5% until the gel is totally submerged.</li>
+
           <li>Put the gel inside the electrophoresis machine and pour TAE 0.5% until the gel is totally submerged.</li>
 
         </ul>
 
         </ul>
 
       <li>Preparation of the samples</li>
 
       <li>Preparation of the samples</li>
 
         <ul>
 
         <ul>
−
           <li>Mix 5uL of each sample with 1uL of 6X loading dye in microcentrifuge tubes/ on a piece of parafilm / in PCR tubes.</li>
+
           <li>Mix 5uL of each sample with 1uL of 6X loading dye in microcentrifuge tubes (or any hydrophobic surface like parafilm) </li>
−
        </ul>
+
 
 
       <li>Pipette 5-6uL of each sample inside a different well.</li>
 
       <li>Pipette 5-6uL of each sample inside a different well.</li>
−
       <li>Pipette 5uL of the appropriate ladder in one of the wells.</li>
+
       <li>Pipette 5uL of the appropriate ladder in one of the wells (Some ladders have a dye in it, but some don't, so one has to add gel loading dye to these ones)</li>
 
       <li>Close the machine and launch the migration with the desired voltage</li>
 
       <li>Close the machine and launch the migration with the desired voltage</li>
−
       <li>After the desired migration time turn of the machine, remove the lid and put the gel inside the Blue light revelator.</li>
+
       <li>After the desired migration time turn off the machine, remove the lid and put the gel inside the gel visualizer.</li>
 +
        </ul>
 
     </ul>
 
     </ul>
 
     </td>
 
     </td>

Latest revision as of 17:12, 6 September 2015

Gel-Electrophoresis protocol
  • Preparation of the Gel (50mL)
    • Mix 0.5 grams of agarose with 50mL 0.5% TAE solution
    • Heat the Erlenmeyer (conical flask) in a microwave until the liquid becomes totally transparent.
    • Let it rest until you can take the Erlenmeyer in your hand without pain for 10 seconds.
    • Add 5uL of SYBR® Safe (10000X) and mix gently until the solution homogenizes.
    • Pour the liquid in a gel mold with the desired number of wells and wait 20-30 min (or until the gel is solidified).
    • Put the gel inside the electrophoresis machine and pour TAE 0.5% until the gel is totally submerged.
  • Preparation of the samples
    • Mix 5uL of each sample with 1uL of 6X loading dye in microcentrifuge tubes (or any hydrophobic surface like parafilm)
    • Pipette 5-6uL of each sample inside a different well.
    • Pipette 5uL of the appropriate ladder in one of the wells (Some ladders have a dye in it, but some don't, so one has to add gel loading dye to these ones)
    • Close the machine and launch the migration with the desired voltage
    • After the desired migration time turn off the machine, remove the lid and put the gel inside the gel visualizer.