http://2012hs.igem.org/wiki/index.php?title=Team:Tyngsboro_MA_Tigers/Results&feed=atom&action=historyTeam:Tyngsboro MA Tigers/Results - Revision history2024-03-29T12:14:36ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012hs.igem.org/wiki/index.php?title=Team:Tyngsboro_MA_Tigers/Results&diff=9276&oldid=prevRravgiala: /* Circuit 1: Proof of Concept */2012-06-17T01:26:02Z<p><span class="autocomment">Circuit 1: Proof of Concept</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 microliters of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other two tubes. Incubate the tubes for 10 minutes at room temperature.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 microliters of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other two tubes. Incubate the tubes for 10 minutes at room temperature.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>12. Tap the closed tubes with your finger to mix. <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>12. Tap the closed tubes with your finger to mix. <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>13. Pipet 100 microliters of the transformation suspension +K145201/+J45200 onto the LB/amp/kan (+) BBa_J45999 <del class="diffchange diffchange-inline">using a new sterile pipet</del>. Repeat this step<del class="diffchange diffchange-inline">, using new sterile pipets, for </del>the LB/amp/kan (-) BBa_J45999 and LB (-) BBa_J45999 plates. <del class="diffchange diffchange-inline"><br></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>13. Pipet 100 microliters of the transformation suspension +K145201/+J45200 onto the LB/amp/kan (+) BBa_J45999 <ins class="diffchange diffchange-inline">plate</ins>. Repeat this step <ins class="diffchange diffchange-inline">by pipetting 100 μl of the -K145201/+J45200 BBa_J45999 onto </ins>the LB/amp/kan (-) BBa_J45999 and LB (-) BBa_J45999 plates.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>14<del class="diffchange diffchange-inline">. Pipet 100 μl of the control suspension (-K145201/+J45200) onto the LB (–) plate, using a new sterile pipet. Repeat for NEB 10-beta cells.<br></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>14. Spread the suspensions evenly around the surface of the agar using the flat surface of a sterile loop. Use a new sterile loop for each plate.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">15</del>. Spread the suspensions evenly around the surface of the agar using the flat surface of a sterile loop. Use a new sterile loop for each plate.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">15</ins>. Stack up your plates and tape them together. Put the stack upside down in the incubator at 37°C until the next day.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">16</del>. Stack up your plates and tape them together. Put the stack upside down in the incubator at 37°C until the next day.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== <B>Circuit 2: Carbon Monoxide Detector</B> ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== <B>Circuit 2: Carbon Monoxide Detector</B> ==</div></td></tr>
</table>Rravgialahttp://2012hs.igem.org/wiki/index.php?title=Team:Tyngsboro_MA_Tigers/Results&diff=9226&oldid=prevRravgiala: /* Circuit 1: Proof of Concept */2012-06-17T01:15:19Z<p><span class="autocomment">Circuit 1: Proof of Concept</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><u>Transformation</u><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><u>Transformation</u><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Remove plates from the incubator. These plates are LB/amp (+) BBa_J45999 and LB/amp (+) NEB 10-beta produced from the previous transformation.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Remove plates from the incubator. These plates are LB/amp (+) BBa_J45999 and LB/amp (+) NEB 10-beta produced from the previous transformation.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>2. Label one tube +K145201/<del class="diffchange diffchange-inline">J45200 BBa_J45999, one tube </del>+J45200 BBa_J45999<del class="diffchange diffchange-inline">, </del>and one tube -J45200 BBa_J45999. Label one more tube +K145201/<del class="diffchange diffchange-inline">J45200 NEB10, one </del>+J45200 NEB10<del class="diffchange diffchange-inline">, </del>and one <del class="diffchange diffchange-inline">tube </del>-J45200 NEB10.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>2. Label one tube +K145201/+J45200 BBa_J45999 and one tube -<ins class="diffchange diffchange-inline">K145201/+</ins>J45200 BBa_J45999. Label one more tube +K145201/+J45200 NEB10 and one -<ins class="diffchange diffchange-inline">K145201/+</ins>J45200 NEB10.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. Open the tubes and, using a sterile transfer pipet, transfer 250 microliters of transformation solution (CaCl2).<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. Open the tubes and, using a sterile transfer pipet, transfer 250 microliters of transformation solution (CaCl2).<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>4. Place the tubes on ice.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>4. Place the tubes on ice.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>5. Use a sterile loop to pick up a single colony of bacteria from the LB/amp (+) BBa_J45999 competent cells plate. Pick up the <del class="diffchange diffchange-inline">BBa_K145201</del>/J45200 tube and immerse the loop in the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tubes back in the tube rack in the ice. Using a new sterile loop, repeat for the +J45200 tube.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>5. Use a sterile loop to pick up a single colony of bacteria from the LB/amp (+) BBa_J45999 competent cells plate. Pick up the <ins class="diffchange diffchange-inline"> +K145201</ins>/<ins class="diffchange diffchange-inline">+</ins>J45200 <ins class="diffchange diffchange-inline">BBa_J45999 </ins>tube and immerse the loop in the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tubes back in the tube rack in the ice. Using a new sterile loop, repeat for the <ins class="diffchange diffchange-inline">-K145201/</ins>+J45200 <ins class="diffchange diffchange-inline">BBa_J45999 </ins>tube.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>6. Immerse a new sterile loop into the stock tube containing the re-suspending K145201 plasmid. Withdraw a loopful. There should be a film of plasmid solution across the ring. Mix the loopful into the cell suspension of the +K145201/J45200 BBa_J45999 tube. Close the tube and return it to the rack on ice. Also close the +J45200 BBa_J45999 tube. Do not add plasmid DNA to <del class="diffchange diffchange-inline">the -J45200</del>. Repeat for LB/amp (+) NEB 10-beta cells.<br> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>6. Immerse a new sterile loop into the stock tube containing the re-suspending K145201 plasmid. Withdraw a loopful. There should be a film of plasmid solution across the ring. Mix the loopful into the cell suspension of the +K145201/<ins class="diffchange diffchange-inline">+</ins>J45200 BBa_J45999 tube. Close the tube and return it to the rack on ice. Also close the <ins class="diffchange diffchange-inline">-K145201/</ins>+J45200 BBa_J45999 tube. Do not add plasmid DNA to <ins class="diffchange diffchange-inline">this tube</ins>. Repeat for LB/amp (+) NEB 10-beta cells.<br> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. Incubate the tubes on ice for 10 minutes. Make sure to push the tube all the way down in the rack so the bottoms of the tubes stick out and make contact with the ice.<br> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. Incubate the tubes on ice for 10 minutes. Make sure to push the tube all the way down in the rack so the bottoms of the tubes stick out and make contact with the ice.<br> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>8. While the tubes are sitting on ice, label your LB nutrient agar plates on the bottom sides as follows:<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>8. While the tubes are sitting on ice, label your LB nutrient agar plates on the bottom sides as follows:<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center>LB/amp/kan (+) BBa_J45999 <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center>LB/amp/kan (+) BBa_J45999 <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB/amp (<del class="diffchange diffchange-inline">+</del>) BBa_J45999 <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB/amp<ins class="diffchange diffchange-inline">/kan </ins>(<ins class="diffchange diffchange-inline">-</ins>) BBa_J45999 <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB (–) BBa_J45999 <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB (–) BBa_J45999 <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp/kan (+) NEB10 <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp/kan (+) NEB10 <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB/amp (<del class="diffchange diffchange-inline">+</del>) NEB10 <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB/amp<ins class="diffchange diffchange-inline">/kan </ins>(<ins class="diffchange diffchange-inline">-</ins>) NEB10 <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB (–) NEB10 </center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB (–) NEB10 </center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>9. Using the foam rack as a holder, transfer the three tubes into the water bath set at 42° C, for exactly 90 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>9. Using the foam rack as a holder, transfer the three tubes into the water bath set at 42° C, for exactly 90 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 microliters of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other two tubes. Incubate the tubes for 10 minutes at room temperature.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 microliters of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other two tubes. Incubate the tubes for 10 minutes at room temperature.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>12. Tap the closed tubes with your finger to mix. <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>12. Tap the closed tubes with your finger to mix. <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>13. Pipet 100 microliters of the transformation suspension +K145201/J45200 onto the LB/amp/kan + BBa_J45999 using a new sterile pipet. Repeat this step, using new sterile pipets, for the LB/amp (<del class="diffchange diffchange-inline">+</del>) BBa_J45999 and LB (-) BBa_J45999 plates. <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>13. Pipet 100 microliters of the transformation suspension +K145201/<ins class="diffchange diffchange-inline">+</ins>J45200 onto the LB/amp/kan <ins class="diffchange diffchange-inline">(</ins>+<ins class="diffchange diffchange-inline">) </ins>BBa_J45999 using a new sterile pipet. Repeat this step, using new sterile pipets, for the LB/amp<ins class="diffchange diffchange-inline">/kan </ins>(<ins class="diffchange diffchange-inline">-</ins>) BBa_J45999 and LB (-) BBa_J45999 plates. <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>14. Pipet 100 μl of the control suspension (-J45200) onto the LB – plate, using a new sterile pipet. Repeat for NEB 10-beta cells.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>14. Pipet 100 μl of the control suspension (-<ins class="diffchange diffchange-inline">K145201/+</ins>J45200) onto the LB <ins class="diffchange diffchange-inline">(</ins>–<ins class="diffchange diffchange-inline">) </ins>plate, using a new sterile pipet. Repeat for NEB 10-beta cells.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>15. Spread the suspensions evenly around the surface of the agar using the flat surface of a sterile loop. Use a new sterile loop for each plate.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>15. Spread the suspensions evenly around the surface of the agar using the flat surface of a sterile loop. Use a new sterile loop for each plate.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>16. Stack up your plates and tape them together. Put the stack upside down in the incubator at 37°C until the next day.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>16. Stack up your plates and tape them together. Put the stack upside down in the incubator at 37°C until the next day.</div></td></tr>
</table>Rravgialahttp://2012hs.igem.org/wiki/index.php?title=Team:Tyngsboro_MA_Tigers/Results&diff=8271&oldid=prevRravgiala: /* Circuit 1: Proof of Concept */2012-06-16T20:18:21Z<p><span class="autocomment">Circuit 1: Proof of Concept</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. After incubating they can be stored in a fridge at 4°C until a culture can be grown.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. After incubating they can be stored in a fridge at 4°C until a culture can be grown.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b>Transformation of Plasmid 1 (BBa_J45200)</B></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b>Transformation of Plasmid 1 (BBa_J45200)</B<ins class="diffchange diffchange-inline">><br</ins>></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><U>Preparing BBa_J45999 and NEB 10-beta cells (competent cells)</U></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><U>Preparing BBa_J45999 and NEB 10-beta cells (competent cells)</U></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>Rravgialahttp://2012hs.igem.org/wiki/index.php?title=Team:Tyngsboro_MA_Tigers/Results&diff=8270&oldid=prevRravgiala: /* Circuit 1: Proof of Concept */2012-06-16T20:17:52Z<p><span class="autocomment">Circuit 1: Proof of Concept</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. After incubating they can be stored in a fridge at 4°C until a culture can be grown.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. After incubating they can be stored in a fridge at 4°C until a culture can be grown.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><b>Transformation of Plasmid 1 (BBa_J45200)</B></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><U>Preparing BBa_J45999 and NEB 10-beta cells (competent cells)</U></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><U>Preparing BBa_J45999 and NEB 10-beta cells (competent cells)</U></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>15. Stack up your plates and tape them together. Put the stack upside down in the incubator at 37°C until the next day.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>15. Stack up your plates and tape them together. Put the stack upside down in the incubator at 37°C until the next day.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><<del class="diffchange diffchange-inline">U</del>>Transformation of <del class="diffchange diffchange-inline">Second </del>Plasmid</<del class="diffchange diffchange-inline">U</del>><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">b</ins>>Transformation of Plasmid <ins class="diffchange diffchange-inline">2 (BBa_K145201)</ins></<ins class="diffchange diffchange-inline">b</ins>><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><u>Make competent cells</u><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><u>Make competent cells</u><br></div></td></tr>
</table>Rravgialahttp://2012hs.igem.org/wiki/index.php?title=Team:Tyngsboro_MA_Tigers/Results&diff=8264&oldid=prevRravgiala: /* Circuit 1: Proof of Concept */2012-06-16T20:13:59Z<p><span class="autocomment">Circuit 1: Proof of Concept</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 20:13, 16 June 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><U>Transformation of Second Plasmid</U><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><U>Transformation of Second Plasmid</U><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">A. </del>Make competent cells<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><u></ins>Make competent cells<ins class="diffchange diffchange-inline"></u></ins><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Remove transformation plate LB/amp (+) BBa_J45999 and LB/amp (+) NEB 10-beta from the incubator or fridge.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Remove transformation plate LB/amp (+) BBa_J45999 and LB/amp (+) NEB 10-beta from the incubator or fridge.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>2. Label one microtube with "NEB 10-beta 2" and one microtube "BBa_J45999" and add 100ul of distilled water to each.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>2. Label one microtube with "NEB 10-beta 2" and one microtube "BBa_J45999" and add 100ul of distilled water to each.<br></div></td></tr>
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<td colspan="2" class="diff-lineno">Line 391:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. Once your agar plate has grown up you can store it in your fridge (4°C) until you're ready to make competent cells.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. Once your agar plate has grown up you can store it in your fridge (4°C) until you're ready to make competent cells.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">B. </del>Re-suspending<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><u></ins>Re-suspending<ins class="diffchange diffchange-inline"></U></ins><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Use a pipette to re-suspend part K145201 with 10 microliters of distilled water. Aspirate up and down a few times to ensure the DNA has been fully re-suspended. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Use a pipette to re-suspend part K145201 with 10 microliters of distilled water. Aspirate up and down a few times to ensure the DNA has been fully re-suspended. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">C. </del>Transformation<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><u></ins>Transformation<ins class="diffchange diffchange-inline"></u></ins><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Remove plates from the incubator. These plates are LB/amp (+) BBa_J45999 and LB/amp (+) NEB 10-beta produced from the previous transformation.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Remove plates from the incubator. These plates are LB/amp (+) BBa_J45999 and LB/amp (+) NEB 10-beta produced from the previous transformation.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>2. Label one tube +K145201/J45200 BBa_J45999, one tube +J45200 BBa_J45999, and one tube -J45200 BBa_J45999. Label one more tube +K145201/J45200 NEB10, one +J45200 NEB10, and one tube -J45200 NEB10.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>2. Label one tube +K145201/J45200 BBa_J45999, one tube +J45200 BBa_J45999, and one tube -J45200 BBa_J45999. Label one more tube +K145201/J45200 NEB10, one +J45200 NEB10, and one tube -J45200 NEB10.<br></div></td></tr>
</table>Rravgialahttp://2012hs.igem.org/wiki/index.php?title=Team:Tyngsboro_MA_Tigers/Results&diff=8261&oldid=prevRravgiala: /* Circuit 1: Proof of Concept */2012-06-16T20:12:25Z<p><span class="autocomment">Circuit 1: Proof of Concept</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 20:12, 16 June 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp (+) NEB 10-beta<br> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp (+) NEB 10-beta<br> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp (–) NEB 10-beta<br> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp (–) NEB 10-beta<br> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB (-) NEB 10-beta</center><br> <del class="diffchange diffchange-inline"> <br> </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB (-) NEB 10-beta</center><br> <ins class="diffchange diffchange-inline"> </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>10. Using the foam rack as a holder, transfer the +J45200 and -J45200 tubes into the water bath set at 42 degrees Celsius, for exactly 90 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>10. Using the foam rack as a holder, transfer the +J45200 and -J45200 tubes into the water bath set at 42 degrees Celsius, for exactly 90 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Directly after the 90 seconds, place both tubes back on ice (0° C). Incubate the tubes on ice for 2 minutes.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Directly after the 90 seconds, place both tubes back on ice (0° C). Incubate the tubes on ice for 2 minutes.<br></div></td></tr>
</table>Rravgialahttp://2012hs.igem.org/wiki/index.php?title=Team:Tyngsboro_MA_Tigers/Results&diff=8260&oldid=prevRravgiala: /* Circuit 1: Proof of Concept */2012-06-16T20:11:51Z<p><span class="autocomment">Circuit 1: Proof of Concept</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
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<col class='diff-content' />
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 20:11, 16 June 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp/kan (+) NEB10 <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp/kan (+) NEB10 <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp (+) NEB10 <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp (+) NEB10 <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB (–) NEB10 <del class="diffchange diffchange-inline"><br></del></center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB (–) NEB10 </center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>9. Using the foam rack as a holder, transfer the three tubes into the water bath set at 42° C, for exactly 90 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>9. Using the foam rack as a holder, transfer the three tubes into the water bath set at 42° C, for exactly 90 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>10. Directly after the 90 seconds, place all three tubes back on ice (0° C). Incubate the tubes on ice for 2 minutes.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>10. Directly after the 90 seconds, place all three tubes back on ice (0° C). Incubate the tubes on ice for 2 minutes.<br></div></td></tr>
</table>Rravgialahttp://2012hs.igem.org/wiki/index.php?title=Team:Tyngsboro_MA_Tigers/Results&diff=8258&oldid=prevRravgiala: /* Circuit 1: Proof of Concept */2012-06-16T20:11:03Z<p><span class="autocomment">Circuit 1: Proof of Concept</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 20:11, 16 June 2012</td>
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<td colspan="2" class="diff-lineno">Line 373:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp (–) NEB 10-beta<br> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp (–) NEB 10-beta<br> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB (-) NEB 10-beta</center><br> <br> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB (-) NEB 10-beta</center><br> <br> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>10. Using the foam rack as a holder, transfer the +J45200 and -J45200 tubes into the water bath set at 42 degrees Celsius, for exactly <del class="diffchange diffchange-inline">50 </del>seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>10. Using the foam rack as a holder, transfer the +J45200 and -J45200 tubes into the water bath set at 42 degrees Celsius, for exactly <ins class="diffchange diffchange-inline">90 </ins>seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Directly after the 90 seconds, place both tubes back on ice (0° C). Incubate the tubes on ice for 2 minutes.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Directly after the 90 seconds, place both tubes back on ice (0° C). Incubate the tubes on ice for 2 minutes.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>12. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 μl of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 minutes at room temperature.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>12. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 μl of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 minutes at room temperature.<br></div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 405:</td>
<td colspan="2" class="diff-lineno">Line 405:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center>LB/amp/kan (+) BBa_J45999 <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center>LB/amp/kan (+) BBa_J45999 <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB/amp<del class="diffchange diffchange-inline">/kan </del>(<del class="diffchange diffchange-inline">-</del>) BBa_J45999 <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB/amp (<ins class="diffchange diffchange-inline">+</ins>) BBa_J45999 <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB (–) BBa_J45999 <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB (–) BBa_J45999 <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp/kan (+) NEB10 <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB/amp/kan (+) NEB10 <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB/amp<del class="diffchange diffchange-inline">/kan </del>(<del class="diffchange diffchange-inline">-</del>) NEB10 <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB/amp (<ins class="diffchange diffchange-inline">+</ins>) NEB10 <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB (–) NEB10 <br></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>LB (–) NEB10 <br></center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>9. Using the foam rack as a holder, transfer the three tubes into the water bath set at 42° C, for exactly 90 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>9. Using the foam rack as a holder, transfer the three tubes into the water bath set at 42° C, for exactly 90 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td></tr>
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<td colspan="2" class="diff-lineno">Line 415:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 microliters of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other two tubes. Incubate the tubes for 10 minutes at room temperature.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 microliters of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other two tubes. Incubate the tubes for 10 minutes at room temperature.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>12. Tap the closed tubes with your finger to mix. <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>12. Tap the closed tubes with your finger to mix. <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>13. Pipet 100 microliters of the transformation suspension +K145201/J45200 onto the LB/amp/kan + using a new sterile pipet. Repeat this step, using new sterile pipets, for the LB+ and LB- plates.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>13. Pipet 100 microliters of the transformation suspension +K145201/J45200 onto the LB/amp/kan + <ins class="diffchange diffchange-inline">BBa_J45999 </ins>using a new sterile pipet. Repeat this step, using new sterile pipets, for the LB<ins class="diffchange diffchange-inline">/amp (</ins>+<ins class="diffchange diffchange-inline">) BBa_J45999 </ins>and LB <ins class="diffchange diffchange-inline">(</ins>-<ins class="diffchange diffchange-inline">) BBa_J45999 </ins>plates. <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>14. Pipet 100 <del class="diffchange diffchange-inline">microliters </del>of the <del class="diffchange diffchange-inline">transformation </del>suspension <del class="diffchange diffchange-inline">+</del>J45200 onto the LB<del class="diffchange diffchange-inline">/amp + </del>using a new sterile pipet. Repeat <del class="diffchange diffchange-inline">this step, using new sterile pipets, </del>for <del class="diffchange diffchange-inline">the LB+ and LB</del>- <del class="diffchange diffchange-inline">plates</del>.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>14. Pipet 100 <ins class="diffchange diffchange-inline">μl </ins>of the <ins class="diffchange diffchange-inline">control </ins>suspension <ins class="diffchange diffchange-inline">(-</ins>J45200<ins class="diffchange diffchange-inline">) </ins>onto the LB <ins class="diffchange diffchange-inline">– plate, </ins>using a new sterile pipet. <ins class="diffchange diffchange-inline"> </ins>Repeat for <ins class="diffchange diffchange-inline">NEB 10</ins>-<ins class="diffchange diffchange-inline">beta cells</ins>.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>15. Spread the suspensions evenly around the surface of the agar using the flat surface of a sterile loop. Use a new sterile loop for each plate.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>15. Spread the suspensions evenly around the surface of the agar using the flat surface of a sterile loop. Use a new sterile loop for each plate.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>16. Stack up your plates and tape them together. Put the stack upside down in the incubator at 37°C until the next day.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>16. Stack up your plates and tape them together. Put the stack upside down in the incubator at 37°C until the next day.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">-----</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><U>Growing up cell cultures</U><br></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">1. Clean the lab bench by wiping down with 70% ethanol and paper towels.<br></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">2. Remove the agar plates from the incubator or fridge.<br></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">3. Label one 14 mL culture tube for BBa_J45999. Label one 14 mL culture tube for NEB-10. Add 5 mL of LB broth to each culture tube.<br></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">4. Use an inoculating loop to pick a single colony from each agar plate and inoculate the LB broth. Press lightly on the snap cap of the 14 mL tube, the capes should be a bit loose to allow for air flow.<br></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">5. Incubate for 16 hours at 37°C in a rotator or shaker (if this is not possible, place on a magnetic stir plate overnight to provide some vibration). <br></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">6. After incubation, the cell culture should be cloudy. You can now firmly press down on the snap caps to seal the tubes and store the cell culture at 4°C until you’re ready to move onto the next step.</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== <B>Circuit 2: Carbon Monoxide Detector</B> ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== <B>Circuit 2: Carbon Monoxide Detector</B> ==</div></td></tr>
</table>Rravgialahttp://2012hs.igem.org/wiki/index.php?title=Team:Tyngsboro_MA_Tigers/Results&diff=8206&oldid=prevRravgiala: /* Circuit 1: Proof of Concept */2012-06-16T19:48:24Z<p><span class="autocomment">Circuit 1: Proof of Concept</span></p>
<table style="background-color: white; color:black;">
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 19:48, 16 June 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>8. While the tubes are sitting on ice, label your LB nutrient agar plates on the bottom sides as follows:<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>8. While the tubes are sitting on ice, label your LB nutrient agar plates on the bottom sides as follows:<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB/amp/kan + BBa_J45999 <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><center></ins>LB/amp/kan <ins class="diffchange diffchange-inline">(</ins>+<ins class="diffchange diffchange-inline">) </ins>BBa_J45999 <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB/amp <del class="diffchange diffchange-inline">+ </del>BBa_J45999 <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB/amp<ins class="diffchange diffchange-inline">/kan (-) </ins>BBa_J45999 <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB – BBa_J45999 <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB <ins class="diffchange diffchange-inline">(</ins>–<ins class="diffchange diffchange-inline">) </ins>BBa_J45999 <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB/amp/kan + NEB10 <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB/amp/kan <ins class="diffchange diffchange-inline">(</ins>+<ins class="diffchange diffchange-inline">) </ins>NEB10 <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB/amp <del class="diffchange diffchange-inline">+ </del>NEB10 <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB/amp<ins class="diffchange diffchange-inline">/kan (-) </ins>NEB10 <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB – NEB10 <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB <ins class="diffchange diffchange-inline">(</ins>–<ins class="diffchange diffchange-inline">) </ins>NEB10 <br<ins class="diffchange diffchange-inline">></center</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>9. Using the foam rack as a holder, transfer the three tubes into the water bath set at 42° C, for exactly <del class="diffchange diffchange-inline">50 </del>seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>9. Using the foam rack as a holder, transfer the three tubes into the water bath set at 42° C, for exactly <ins class="diffchange diffchange-inline">90 </ins>seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>10. Directly after the <del class="diffchange diffchange-inline">50 </del>seconds, place all three tubes back on ice (0° C). Incubate the tubes on ice for 2 minutes.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>10. Directly after the <ins class="diffchange diffchange-inline">90 </ins>seconds, place all three tubes back on ice (0° C). Incubate the tubes on ice for 2 minutes.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 microliters of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other two tubes. Incubate the tubes for 10 minutes at room temperature.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 microliters of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other two tubes. Incubate the tubes for 10 minutes at room temperature.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>12. Tap the closed tubes with your finger to mix. <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>12. Tap the closed tubes with your finger to mix. <br></div></td></tr>
</table>Rravgialahttp://2012hs.igem.org/wiki/index.php?title=Team:Tyngsboro_MA_Tigers/Results&diff=8191&oldid=prevRravgiala: /* Circuit 1: Proof of Concept */2012-06-16T19:44:02Z<p><span class="autocomment">Circuit 1: Proof of Concept</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 19:44, 16 June 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>6. Immerse a new sterile loop into the stock tube containing the re-suspended J45200 plasmid. Withdraw a loopful which is approximately 10μl of DNA.<br> There should be a film of plasmid solution across the ring. Mix the loopful into the cell suspension of the +J45200 tube. Close the tube and return it to the rack on ice. Also close the -J45200 tube. Do not add plasmid DNA to the -J45200. <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>6. Immerse a new sterile loop into the stock tube containing the re-suspended J45200 plasmid. Withdraw a loopful which is approximately 10μl of DNA.<br> There should be a film of plasmid solution across the ring. Mix the loopful into the cell suspension of the +J45200 tube. Close the tube and return it to the rack on ice. Also close the -J45200 tube. Do not add plasmid DNA to the -J45200. <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. Repeat for NEB 10-beta cells.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. Repeat for NEB 10-beta cells.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">7</del>. Incubate the tubes on ice for 10 minutes. Make sure to push the tube all the way down in the rack so the bottoms of the tubes stick out and make contact with the ice. <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">8</del>. While the tubes are sitting on ice, label your 6 LB nutrient agar plates on the bottom sides as follows:<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">8</ins>. Incubate the tubes on ice for 10 minutes. Make sure to push the tube all the way down in the rack so the bottoms of the tubes stick out and make contact with the ice. <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><center>LB/amp (+) BBBa_J45999 <br> <del class="diffchange diffchange-inline">LB/amp (+) NEB 10-beta<br></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">9</ins>. While the tubes are sitting on ice, label your 6 LB nutrient agar plates on the bottom sides as follows:<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB/amp (–) BBa_J45999 <br> LB/amp (<del class="diffchange diffchange-inline">–</del>) NEB 10-beta<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><center>LB/amp (+) BBBa_J45999 <br> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB (<del class="diffchange diffchange-inline">-</del>) <del class="diffchange diffchange-inline">BBa_J45999 </del><br> <del class="diffchange diffchange-inline"> </del>LB (-) NEB 10-beta</center><br> <del class="diffchange diffchange-inline"> </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB/amp (–) BBa_J45999 <br> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">9</del>. Using the foam rack as a holder, transfer <del class="diffchange diffchange-inline">both </del>the +J45200 and -J45200 tubes into the water bath set at 42 degrees Celsius, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">LB (-) BBa_J45999 </ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">10</del>. Directly after the <del class="diffchange diffchange-inline">50 </del>seconds, place both tubes back on ice (0° C). Incubate the tubes on ice for 2 minutes.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">11</del>. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 <del class="diffchange diffchange-inline">microliters </del>of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 minutes at room temperature.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB/amp (<ins class="diffchange diffchange-inline">+</ins>) NEB 10-beta<br> <ins class="diffchange diffchange-inline"> </ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">12</del>. Tap the closed tubes with your finger to mix. Pipet 100 <del class="diffchange diffchange-inline">microliters </del>of the transformation suspension (+J45200) onto the LB/amp + plate using a new sterile pipet. Pipet 100 <del class="diffchange diffchange-inline">microliters </del>of the control suspension (-J45200) onto the LB/amp – plate, using a new sterile pipet.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB<ins class="diffchange diffchange-inline">/amp </ins>(<ins class="diffchange diffchange-inline">–</ins>) <ins class="diffchange diffchange-inline">NEB 10-beta</ins><br> <ins class="diffchange diffchange-inline"> </ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">13</del>. Spread the suspensions evenly around the surface of the agar using the flat surface of a sterile loop. Use a new sterile loop for each plate.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB (-) NEB 10-beta</center><br> <ins class="diffchange diffchange-inline"> <br> </ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">14</del>. Stack up your plates and tape them together. Put the stack upside down in the incubator at 37°C until the next day.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">10</ins>. Using the foam rack as a holder, transfer the +J45200 and -J45200 tubes into the water bath set at 42 degrees Celsius, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">11</ins>. Directly after the <ins class="diffchange diffchange-inline">90 </ins>seconds, place both tubes back on ice (0° C). Incubate the tubes on ice for 2 minutes.<br></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">12</ins>. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 <ins class="diffchange diffchange-inline">μl </ins>of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 minutes at room temperature.<br></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">13</ins>. Tap the closed tubes with your finger to mix. Pipet 100 <ins class="diffchange diffchange-inline">μl </ins>of the transformation suspension (+J45200 <ins class="diffchange diffchange-inline">for BBBa_J45999</ins>) onto the LB/amp + plate using a new sterile pipet. Pipet 100 <ins class="diffchange diffchange-inline">μl </ins>of the control suspension (-J45200) onto the LB/amp – plate, using a new sterile pipet<ins class="diffchange diffchange-inline">. Pipet 100 μl of the control suspension (-J45200) onto the LB – plate, using a new sterile pipet. Repeat for NEB 10-beta cells</ins>.<br></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">14</ins>. Spread the suspensions evenly around the surface of the agar using the flat surface of a sterile loop. Use a new sterile loop for each plate.<br></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">15</ins>. Stack up your plates and tape them together. Put the stack upside down in the incubator at 37°C until the next day.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><U>Transformation of Second Plasmid</U><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><U>Transformation of Second Plasmid</U><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A. Make competent cells<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A. Make competent cells<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>1. Remove transformation plate LB/amp + from the incubator or fridge.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>1. Remove transformation plate LB/amp <ins class="diffchange diffchange-inline">(</ins>+<ins class="diffchange diffchange-inline">) BBa_J45999 and LB/amp (+) NEB 10-beta </ins>from the incubator or fridge.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>2. Label one <del class="diffchange diffchange-inline">0.6ml tube </del>with "NEB 10-beta 2" and add 100ul of distilled water to <del class="diffchange diffchange-inline">it</del>.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>2. Label one <ins class="diffchange diffchange-inline">microtube </ins>with "NEB 10-beta 2<ins class="diffchange diffchange-inline">" and one microtube "BBa_J45999</ins>" and add 100ul of distilled water to <ins class="diffchange diffchange-inline">each</ins>.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>3. Use an inoculating loop, or pipette tip, to pick a single colony from the LB/amp + plate and inoculate the distilled water.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>3. Use an inoculating loop, or pipette tip, to pick a single colony from the LB/amp + <ins class="diffchange diffchange-inline"> </ins>plate and inoculate the distilled water <ins class="diffchange diffchange-inline">for each cell type</ins>.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>4. Pipette up and down to re-suspend the colony into the distilled water, and transfer to the fresh <del class="diffchange diffchange-inline">SOB </del>agar <del class="diffchange diffchange-inline">plate</del>.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>4. Pipette up and down to re-suspend the colony into the distilled water, and transfer to the fresh <ins class="diffchange diffchange-inline">LB/amp </ins>agar <ins class="diffchange diffchange-inline">plates</ins>.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>5. Spread the cells across the plate using either an inoculating loop, or by pouring 3-6 glass beads into the plate, covering, and shaking from side to side.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>5. Spread the cells across the plate using either an inoculating loop, or by pouring 3-6 glass beads into the plate, covering, and shaking from side to side.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>6. Place the <del class="diffchange diffchange-inline">SOB </del>agar plate into the incubator with the agar side facing up, lid facing down (see picture below). Incubate the plate at 37°C for 14-16 hours. Alternately, incubate at room temperature for 24-30 hours.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>6. Place the <ins class="diffchange diffchange-inline">LB/amp </ins>agar plate into the incubator with the agar side facing up, lid facing down (see picture below). Incubate the plate at 37°C for 14-16 hours. Alternately, incubate at room temperature for 24-30 hours.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. Once your agar plate has grown up you can store it in your fridge (4°C) until you're ready to make competent cells.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. Once your agar plate has grown up you can store it in your fridge (4°C) until you're ready to make competent cells.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 390:</td>
<td colspan="2" class="diff-lineno">Line 395:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>C. Transformation<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>C. Transformation<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>1. Remove <del class="diffchange diffchange-inline">plate </del>from the incubator. <del class="diffchange diffchange-inline">This plate is the </del>LB/amp + <del class="diffchange diffchange-inline">sample used to make competent cells</del>.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>1. Remove <ins class="diffchange diffchange-inline">plates </ins>from the incubator. <ins class="diffchange diffchange-inline">These plates are </ins>LB/amp <ins class="diffchange diffchange-inline">(</ins>+<ins class="diffchange diffchange-inline">) BBa_J45999 and LB/amp (+) NEB 10-beta produced from the previous transformation</ins>.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>2. Label one tube +K145201/J45200 and one tube +J45200.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>2. Label one tube +K145201/J45200 <ins class="diffchange diffchange-inline">BBa_J45999, one tube +J45200 BBa_J45999, </ins>and one <ins class="diffchange diffchange-inline">tube -J45200 BBa_J45999. Label one more </ins>tube +<ins class="diffchange diffchange-inline">K145201/J45200 NEB10, one +J45200 NEB10, and one tube -</ins>J45200 <ins class="diffchange diffchange-inline">NEB10</ins>.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. Open the tubes and, using a sterile transfer pipet, transfer 250 microliters of transformation solution (CaCl2).<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. Open the tubes and, using a sterile transfer pipet, transfer 250 microliters of transformation solution (CaCl2).<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>4. Place the tubes on ice.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>4. Place the tubes on ice.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>5. Use a sterile loop to pick up a single colony of bacteria from the LB/amp + competent cells plate. Pick up the <del class="diffchange diffchange-inline">+K145201 </del>and immerse the loop in the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tubes back in the tube rack in the ice. Using a new sterile loop, repeat for the +J45200 tube.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>5. Use a sterile loop to pick up a single colony of bacteria from the LB/amp <ins class="diffchange diffchange-inline">(</ins>+<ins class="diffchange diffchange-inline">) BBa_J45999 </ins>competent cells plate. Pick up the <ins class="diffchange diffchange-inline">BBa_K145201/J45200 tube </ins>and immerse the loop in the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tubes back in the tube rack in the ice. Using a new sterile loop, repeat for the +J45200 tube.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>6. Immerse a new sterile loop into the stock tube containing the re-suspending K145201 plasmid. Withdraw a loopful. There should be a film of plasmid solution across the ring. Mix the loopful into the cell suspension of the +K145201/J45200 tube. Close the tube and return it to the rack on ice. Also close the +J45200 tube. Do not add plasmid DNA to the -J45200.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>6. Immerse a new sterile loop into the stock tube containing the re-suspending K145201 plasmid. Withdraw a loopful. There should be a film of plasmid solution across the ring. Mix the loopful into the cell suspension of the +K145201/J45200 <ins class="diffchange diffchange-inline">BBa_J45999 </ins>tube. Close the tube and return it to the rack on ice. Also close the +J45200 <ins class="diffchange diffchange-inline">BBa_J45999 </ins>tube. Do not add plasmid DNA to the -J45200<ins class="diffchange diffchange-inline">. Repeat for LB/amp (+) NEB 10-beta cells</ins>.<br> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. Incubate the tubes on ice for 10 minutes. Make sure to push the tube all the way down in the rack so the bottoms of the tubes stick out and make contact with the ice.<br> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7. Incubate the tubes on ice for 10 minutes. Make sure to push the tube all the way down in the rack so the bottoms of the tubes stick out and make contact with the ice.<br> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>8. While the tubes are sitting on ice, label your LB nutrient agar plates on the bottom sides as follows:<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>8. While the tubes are sitting on ice, label your LB nutrient agar plates on the bottom sides as follows:<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB/amp/kan +<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB/amp +<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB/amp/kan + <ins class="diffchange diffchange-inline">BBa_J45999 </ins><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB +<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB/amp + <ins class="diffchange diffchange-inline">BBa_J45999 </ins><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>LB –<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB <ins class="diffchange diffchange-inline">– BBa_J45999 <br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">LB/amp/kan + NEB10 <br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">LB/amp </ins>+ <ins class="diffchange diffchange-inline">NEB10 </ins><br></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>LB – <ins class="diffchange diffchange-inline">NEB10 </ins><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>9. Using the foam rack as a holder, transfer the three tubes into the water bath set at 42° C, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>9. Using the foam rack as a holder, transfer the three tubes into the water bath set at 42° C, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>10. Directly after the 50 seconds, place all three tubes back on ice (0° C). Incubate the tubes on ice for 2 minutes.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>10. Directly after the 50 seconds, place all three tubes back on ice (0° C). Incubate the tubes on ice for 2 minutes.<br></div></td></tr>
</table>Rravgiala