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<h2>Protocols</h2> | <h2>Protocols</h2> | ||
<h6>Here we can talk about protocols in the lab</h6> | <h6>Here we can talk about protocols in the lab</h6> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <h3>Transformation with Digest</h3> | ||
+ | <div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6> | ||
+ | <ul> | ||
+ | <li>DNA miniprrep</li> | ||
+ | <li>LB agar plates, Cat No. Teknova, appropriate antibiotic</li> | ||
+ | <li>DH5a competent cells, Invitrogen, Cat 18265-017</li> | ||
+ | <li>SOC (Recovery Medium), Lucigen, Cat No. F98226</li> | ||
+ | <li>1.5 mL tube</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Pipet and tips</li> | ||
+ | <li>Ice bucket and ice</li> | ||
+ | <li>Water bath (42°C)</li> | ||
+ | <li>Incubator (37°C) and shaker</li> | ||
+ | </ul> | ||
+ | </h6> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMethods"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMethods-collapse" aria-expanded="false" aria-controls="transformationDigestMethods-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMethods"> | ||
+ | <div class="panel-body"> | ||
+ | <center><h6> | ||
+ | <ol type="a"> | ||
+ | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>Turn on incubator-shaker at 37°C.</li> | ||
+ | <li>Turn on incubator for plates at 37°C.</li> | ||
+ | <li>Set up water bath at 42°C.</li> | ||
+ | <li>Bring to room temperature S.O.C medium.</li> | ||
+ | <li>Bring LB plates supplemented with appropriate antibiotic at room temperature.</li> | ||
+ | <li>Thaw competent cells on ice.</li> | ||
+ | <li>Aliquots competent cells in as many tubes as needed.</li> | ||
+ | <li>Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix</li> | ||
+ | <li>Incubate on ice for 20 minutes</li> | ||
+ | <li>Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.</li> | ||
+ | <li>Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube</li> | ||
+ | <li>Incubate in shaker at 37°C, 225 rpm for 30 min</li> | ||
+ | <li>Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)</li> | ||
+ | <li>Incubate plates at 37°C overnight</li> | ||
+ | <li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | ||
+ | |||
+ | |||
+ | |||
+ | </ol></h6></center> | ||
+ | <br> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div></div> | ||
+ | |||
+ | <h3>Transformation with Digest</h3> | ||
+ | <div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6> | ||
+ | <ul> | ||
+ | <li>DNA miniprrep</li> | ||
+ | <li>LB agar plates, Cat No. Teknova, appropriate antibiotic</li> | ||
+ | <li>DH5a competent cells, Invitrogen, Cat 18265-017</li> | ||
+ | <li>SOC (Recovery Medium), Lucigen, Cat No. F98226</li> | ||
+ | <li>1.5 mL tube</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Pipet and tips</li> | ||
+ | <li>Ice bucket and ice</li> | ||
+ | <li>Water bath (42°C)</li> | ||
+ | <li>Incubator (37°C) and shaker</li> | ||
+ | </ul> | ||
+ | </h6> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMethods"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMethods-collapse" aria-expanded="false" aria-controls="transformationDigestMethods-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMethods"> | ||
+ | <div class="panel-body"> | ||
+ | <center><h6> | ||
+ | <ol type="a"> | ||
+ | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>Turn on incubator-shaker at 37°C.</li> | ||
+ | <li>Turn on incubator for plates at 37°C.</li> | ||
+ | <li>Set up water bath at 42°C.</li> | ||
+ | <li>Bring to room temperature S.O.C medium.</li> | ||
+ | <li>Bring LB plates supplemented with appropriate antibiotic at room temperature.</li> | ||
+ | <li>Thaw competent cells on ice.</li> | ||
+ | <li>Aliquots competent cells in as many tubes as needed.</li> | ||
+ | <li>Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix</li> | ||
+ | <li>Incubate on ice for 20 minutes</li> | ||
+ | <li>Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.</li> | ||
+ | <li>Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube</li> | ||
+ | <li>Incubate in shaker at 37°C, 225 rpm for 30 min</li> | ||
+ | <li>Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)</li> | ||
+ | <li>Incubate plates at 37°C overnight</li> | ||
+ | <li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | ||
+ | |||
+ | |||
+ | |||
+ | </ol></h6></center> | ||
+ | <br> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div></div><h3>Transformation with Digest</h3> | ||
+ | <div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6> | ||
+ | <ul> | ||
+ | <li>DNA miniprrep</li> | ||
+ | <li>LB agar plates, Cat No. Teknova, appropriate antibiotic</li> | ||
+ | <li>DH5a competent cells, Invitrogen, Cat 18265-017</li> | ||
+ | <li>SOC (Recovery Medium), Lucigen, Cat No. F98226</li> | ||
+ | <li>1.5 mL tube</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Pipet and tips</li> | ||
+ | <li>Ice bucket and ice</li> | ||
+ | <li>Water bath (42°C)</li> | ||
+ | <li>Incubator (37°C) and shaker</li> | ||
+ | </ul> | ||
+ | </h6> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMethods"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMethods-collapse" aria-expanded="false" aria-controls="transformationDigestMethods-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMethods"> | ||
+ | <div class="panel-body"> | ||
+ | <center><h6> | ||
+ | <ol type="a"> | ||
+ | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>Turn on incubator-shaker at 37°C.</li> | ||
+ | <li>Turn on incubator for plates at 37°C.</li> | ||
+ | <li>Set up water bath at 42°C.</li> | ||
+ | <li>Bring to room temperature S.O.C medium.</li> | ||
+ | <li>Bring LB plates supplemented with appropriate antibiotic at room temperature.</li> | ||
+ | <li>Thaw competent cells on ice.</li> | ||
+ | <li>Aliquots competent cells in as many tubes as needed.</li> | ||
+ | <li>Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix</li> | ||
+ | <li>Incubate on ice for 20 minutes</li> | ||
+ | <li>Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.</li> | ||
+ | <li>Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube</li> | ||
+ | <li>Incubate in shaker at 37°C, 225 rpm for 30 min</li> | ||
+ | <li>Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)</li> | ||
+ | <li>Incubate plates at 37°C overnight</li> | ||
+ | <li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | ||
+ | |||
+ | |||
+ | |||
+ | </ol></h6></center> | ||
+ | <br> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div></div> | ||
+ | |||
+ | |||
+ | <h3>Transformation with Digest</h3> | ||
+ | <div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6> | ||
+ | <ul> | ||
+ | <li>DNA miniprrep</li> | ||
+ | <li>LB agar plates, Cat No. Teknova, appropriate antibiotic</li> | ||
+ | <li>DH5a competent cells, Invitrogen, Cat 18265-017</li> | ||
+ | <li>SOC (Recovery Medium), Lucigen, Cat No. F98226</li> | ||
+ | <li>1.5 mL tube</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Pipet and tips</li> | ||
+ | <li>Ice bucket and ice</li> | ||
+ | <li>Water bath (42°C)</li> | ||
+ | <li>Incubator (37°C) and shaker</li> | ||
+ | </ul> | ||
+ | </h6> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMethods"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMethods-collapse" aria-expanded="false" aria-controls="transformationDigestMethods-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMethods"> | ||
+ | <div class="panel-body"> | ||
+ | <center><h6> | ||
+ | <ol type="a"> | ||
+ | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>Turn on incubator-shaker at 37°C.</li> | ||
+ | <li>Turn on incubator for plates at 37°C.</li> | ||
+ | <li>Set up water bath at 42°C.</li> | ||
+ | <li>Bring to room temperature S.O.C medium.</li> | ||
+ | <li>Bring LB plates supplemented with appropriate antibiotic at room temperature.</li> | ||
+ | <li>Thaw competent cells on ice.</li> | ||
+ | <li>Aliquots competent cells in as many tubes as needed.</li> | ||
+ | <li>Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix</li> | ||
+ | <li>Incubate on ice for 20 minutes</li> | ||
+ | <li>Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.</li> | ||
+ | <li>Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube</li> | ||
+ | <li>Incubate in shaker at 37°C, 225 rpm for 30 min</li> | ||
+ | <li>Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)</li> | ||
+ | <li>Incubate plates at 37°C overnight</li> | ||
+ | <li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | ||
+ | |||
+ | |||
+ | |||
+ | </ol></h6></center> | ||
+ | <br> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div></div> | ||
+ | <h3>Transformation with Digest</h3> | ||
+ | <div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6> | ||
+ | <ul> | ||
+ | <li>DNA miniprrep</li> | ||
+ | <li>LB agar plates, Cat No. Teknova, appropriate antibiotic</li> | ||
+ | <li>DH5a competent cells, Invitrogen, Cat 18265-017</li> | ||
+ | <li>SOC (Recovery Medium), Lucigen, Cat No. F98226</li> | ||
+ | <li>1.5 mL tube</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Pipet and tips</li> | ||
+ | <li>Ice bucket and ice</li> | ||
+ | <li>Water bath (42°C)</li> | ||
+ | <li>Incubator (37°C) and shaker</li> | ||
+ | </ul> | ||
+ | </h6> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMethods"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMethods-collapse" aria-expanded="false" aria-controls="transformationDigestMethods-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMethods"> | ||
+ | <div class="panel-body"> | ||
+ | <center><h6> | ||
+ | <ol type="a"> | ||
+ | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>Turn on incubator-shaker at 37°C.</li> | ||
+ | <li>Turn on incubator for plates at 37°C.</li> | ||
+ | <li>Set up water bath at 42°C.</li> | ||
+ | <li>Bring to room temperature S.O.C medium.</li> | ||
+ | <li>Bring LB plates supplemented with appropriate antibiotic at room temperature.</li> | ||
+ | <li>Thaw competent cells on ice.</li> | ||
+ | <li>Aliquots competent cells in as many tubes as needed.</li> | ||
+ | <li>Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix</li> | ||
+ | <li>Incubate on ice for 20 minutes</li> | ||
+ | <li>Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.</li> | ||
+ | <li>Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube</li> | ||
+ | <li>Incubate in shaker at 37°C, 225 rpm for 30 min</li> | ||
+ | <li>Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)</li> | ||
+ | <li>Incubate plates at 37°C overnight</li> | ||
+ | <li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | ||
+ | |||
+ | |||
+ | |||
+ | </ol></h6></center> | ||
+ | <br> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div></div><h3>Transformation with Digest</h3> | ||
+ | <div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6> | ||
+ | <ul> | ||
+ | <li>DNA miniprrep</li> | ||
+ | <li>LB agar plates, Cat No. Teknova, appropriate antibiotic</li> | ||
+ | <li>DH5a competent cells, Invitrogen, Cat 18265-017</li> | ||
+ | <li>SOC (Recovery Medium), Lucigen, Cat No. F98226</li> | ||
+ | <li>1.5 mL tube</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Pipet and tips</li> | ||
+ | <li>Ice bucket and ice</li> | ||
+ | <li>Water bath (42°C)</li> | ||
+ | <li>Incubator (37°C) and shaker</li> | ||
+ | </ul> | ||
+ | </h6> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMethods"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMethods-collapse" aria-expanded="false" aria-controls="transformationDigestMethods-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMethods"> | ||
+ | <div class="panel-body"> | ||
+ | <center><h6> | ||
+ | <ol type="a"> | ||
+ | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>Turn on incubator-shaker at 37°C.</li> | ||
+ | <li>Turn on incubator for plates at 37°C.</li> | ||
+ | <li>Set up water bath at 42°C.</li> | ||
+ | <li>Bring to room temperature S.O.C medium.</li> | ||
+ | <li>Bring LB plates supplemented with appropriate antibiotic at room temperature.</li> | ||
+ | <li>Thaw competent cells on ice.</li> | ||
+ | <li>Aliquots competent cells in as many tubes as needed.</li> | ||
+ | <li>Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix</li> | ||
+ | <li>Incubate on ice for 20 minutes</li> | ||
+ | <li>Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.</li> | ||
+ | <li>Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube</li> | ||
+ | <li>Incubate in shaker at 37°C, 225 rpm for 30 min</li> | ||
+ | <li>Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)</li> | ||
+ | <li>Incubate plates at 37°C overnight</li> | ||
+ | <li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | ||
+ | |||
+ | |||
+ | |||
+ | </ol></h6></center> | ||
+ | <br> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div></div><h3>Transformation with Digest</h3> | ||
+ | <div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6> | ||
+ | <ul> | ||
+ | <li>DNA miniprrep</li> | ||
+ | <li>LB agar plates, Cat No. Teknova, appropriate antibiotic</li> | ||
+ | <li>DH5a competent cells, Invitrogen, Cat 18265-017</li> | ||
+ | <li>SOC (Recovery Medium), Lucigen, Cat No. F98226</li> | ||
+ | <li>1.5 mL tube</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Pipet and tips</li> | ||
+ | <li>Ice bucket and ice</li> | ||
+ | <li>Water bath (42°C)</li> | ||
+ | <li>Incubator (37°C) and shaker</li> | ||
+ | </ul> | ||
+ | </h6> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMethods"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMethods-collapse" aria-expanded="false" aria-controls="transformationDigestMethods-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMethods"> | ||
+ | <div class="panel-body"> | ||
+ | <center><h6> | ||
+ | <ol type="a"> | ||
+ | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>Turn on incubator-shaker at 37°C.</li> | ||
+ | <li>Turn on incubator for plates at 37°C.</li> | ||
+ | <li>Set up water bath at 42°C.</li> | ||
+ | <li>Bring to room temperature S.O.C medium.</li> | ||
+ | <li>Bring LB plates supplemented with appropriate antibiotic at room temperature.</li> | ||
+ | <li>Thaw competent cells on ice.</li> | ||
+ | <li>Aliquots competent cells in as many tubes as needed.</li> | ||
+ | <li>Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix</li> | ||
+ | <li>Incubate on ice for 20 minutes</li> | ||
+ | <li>Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.</li> | ||
+ | <li>Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube</li> | ||
+ | <li>Incubate in shaker at 37°C, 225 rpm for 30 min</li> | ||
+ | <li>Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)</li> | ||
+ | <li>Incubate plates at 37°C overnight</li> | ||
+ | <li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | ||
+ | |||
+ | |||
+ | |||
+ | </ol></h6></center> | ||
+ | <br> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div></div><h3>Transformation with Digest</h3> | ||
+ | <div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6> | ||
+ | <ul> | ||
+ | <li>DNA miniprrep</li> | ||
+ | <li>LB agar plates, Cat No. Teknova, appropriate antibiotic</li> | ||
+ | <li>DH5a competent cells, Invitrogen, Cat 18265-017</li> | ||
+ | <li>SOC (Recovery Medium), Lucigen, Cat No. F98226</li> | ||
+ | <li>1.5 mL tube</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Pipet and tips</li> | ||
+ | <li>Ice bucket and ice</li> | ||
+ | <li>Water bath (42°C)</li> | ||
+ | <li>Incubator (37°C) and shaker</li> | ||
+ | </ul> | ||
+ | </h6> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMethods"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMethods-collapse" aria-expanded="false" aria-controls="transformationDigestMethods-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMethods"> | ||
+ | <div class="panel-body"> | ||
+ | <center><h6> | ||
+ | <ol type="a"> | ||
+ | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>Turn on incubator-shaker at 37°C.</li> | ||
+ | <li>Turn on incubator for plates at 37°C.</li> | ||
+ | <li>Set up water bath at 42°C.</li> | ||
+ | <li>Bring to room temperature S.O.C medium.</li> | ||
+ | <li>Bring LB plates supplemented with appropriate antibiotic at room temperature.</li> | ||
+ | <li>Thaw competent cells on ice.</li> | ||
+ | <li>Aliquots competent cells in as many tubes as needed.</li> | ||
+ | <li>Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix</li> | ||
+ | <li>Incubate on ice for 20 minutes</li> | ||
+ | <li>Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.</li> | ||
+ | <li>Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube</li> | ||
+ | <li>Incubate in shaker at 37°C, 225 rpm for 30 min</li> | ||
+ | <li>Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)</li> | ||
+ | <li>Incubate plates at 37°C overnight</li> | ||
+ | <li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | ||
+ | |||
+ | |||
+ | |||
+ | </ol></h6></center> | ||
+ | <br> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div></div><h3>Transformation with Digest</h3> | ||
+ | <div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6> | ||
+ | <ul> | ||
+ | <li>DNA miniprrep</li> | ||
+ | <li>LB agar plates, Cat No. Teknova, appropriate antibiotic</li> | ||
+ | <li>DH5a competent cells, Invitrogen, Cat 18265-017</li> | ||
+ | <li>SOC (Recovery Medium), Lucigen, Cat No. F98226</li> | ||
+ | <li>1.5 mL tube</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Pipet and tips</li> | ||
+ | <li>Ice bucket and ice</li> | ||
+ | <li>Water bath (42°C)</li> | ||
+ | <li>Incubator (37°C) and shaker</li> | ||
+ | </ul> | ||
+ | </h6> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="transformationDigestMethods"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationDigestMethods-collapse" aria-expanded="false" aria-controls="transformationDigestMethods-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="transformationDigestMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="transformationDigestMethods"> | ||
+ | <div class="panel-body"> | ||
+ | <center><h6> | ||
+ | <ol type="a"> | ||
+ | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
+ | <li>Turn on incubator-shaker at 37°C.</li> | ||
+ | <li>Turn on incubator for plates at 37°C.</li> | ||
+ | <li>Set up water bath at 42°C.</li> | ||
+ | <li>Bring to room temperature S.O.C medium.</li> | ||
+ | <li>Bring LB plates supplemented with appropriate antibiotic at room temperature.</li> | ||
+ | <li>Thaw competent cells on ice.</li> | ||
+ | <li>Aliquots competent cells in as many tubes as needed.</li> | ||
+ | <li>Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix</li> | ||
+ | <li>Incubate on ice for 20 minutes</li> | ||
+ | <li>Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.</li> | ||
+ | <li>Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube</li> | ||
+ | <li>Incubate in shaker at 37°C, 225 rpm for 30 min</li> | ||
+ | <li>Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)</li> | ||
+ | <li>Incubate plates at 37°C overnight</li> | ||
+ | <li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | ||
+ | |||
+ | |||
+ | |||
+ | </ol></h6></center> | ||
+ | <br> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div></div> | ||
+ | |||
+ | |||
Revision as of 17:02, 1 November 2017