Difference between revisions of "Team:TecCEM/Experiments"

Line 227: Line 227:
 
             <div class="dropdown paragraphU paddingButton">
 
             <div class="dropdown paragraphU paddingButton">
 
                 <button class="btn btn-primary dropdown-toggle responsiveImage" type="button" data-toggle= "dropdown">Protocol F
 
                 <button class="btn btn-primary dropdown-toggle responsiveImage" type="button" data-toggle= "dropdown">Protocol F
 +
                <span class="caret"></span></button>
 +
                <div class="dropdown-menu">
 +
                    <h3>Preparation of chemocompetent cells</h3>
 +
                    <ul><h4>Materials</h4>
 +
                        <li>LB Medium</li>
 +
                        <li>01. M CaCI2</li>
 +
                        <li>CaCL2 0.1M + 15% glycerol</li>
 +
                        <li>Sterile Falcon tubes</li>
 +
                    </ul>
 +
                    <ol><h4>Steps</h4>
 +
                        <li>Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.</li>
 +
                        <li>Inoculate 100 mL of LB broth with 1 mL of the previous culture.</li>
 +
                        <li>Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.</li>
 +
                        <li>Place on ice for 10 minutes.</li>
 +
                        <li>Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.</li>
 +
                        <li>Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.</li>
 +
                        <li>Centrifuge at 4000 rpm for 10 minutes.</li>
 +
                        <li>Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.</li>
 +
                        <li>Prepare 200 μl aliquots and store at -80 °C.</li>
 +
                    </ol>
 +
                </div>
 +
            </div>
 +
        </div>
 +
        <div class = "col-md-2"></div>
 +
    </div>
 +
    <div class="row">
 +
        <div class = "col-md-2"></div>
 +
        <div class="col-md-8">
 +
            <div class="dropdown paragraphU paddingButton">
 +
                <button class="btn btn-primary dropdown-toggle responsiveImage" type="button" data-toggle= "dropdown">Protocol G
 +
                <span class="caret"></span></button>
 +
                <div class="dropdown-menu">
 +
                    <h3>Preparation of chemocompetent cells</h3>
 +
                    <ul><h4>Materials</h4>
 +
                        <li>LB Medium</li>
 +
                        <li>01. M CaCI2</li>
 +
                        <li>CaCL2 0.1M + 15% glycerol</li>
 +
                        <li>Sterile Falcon tubes</li>
 +
                    </ul>
 +
                    <ol><h4>Steps</h4>
 +
                        <li>Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.</li>
 +
                        <li>Inoculate 100 mL of LB broth with 1 mL of the previous culture.</li>
 +
                        <li>Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.</li>
 +
                        <li>Place on ice for 10 minutes.</li>
 +
                        <li>Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.</li>
 +
                        <li>Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.</li>
 +
                        <li>Centrifuge at 4000 rpm for 10 minutes.</li>
 +
                        <li>Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.</li>
 +
                        <li>Prepare 200 μl aliquots and store at -80 °C.</li>
 +
                    </ol>
 +
                </div>
 +
            </div>
 +
        </div>
 +
        <div class = "col-md-2"></div>
 +
    </div>
 +
    <div class="row">
 +
        <div class = "col-md-2"></div>
 +
        <div class="col-md-8">
 +
            <div class="dropdown paragraphU paddingButton">
 +
                <button class="btn btn-primary dropdown-toggle responsiveImage" type="button" data-toggle= "dropdown">Protocol H
 +
                <span class="caret"></span></button>
 +
                <div class="dropdown-menu">
 +
                    <h3>Preparation of chemocompetent cells</h3>
 +
                    <ul><h4>Materials</h4>
 +
                        <li>LB Medium</li>
 +
                        <li>01. M CaCI2</li>
 +
                        <li>CaCL2 0.1M + 15% glycerol</li>
 +
                        <li>Sterile Falcon tubes</li>
 +
                    </ul>
 +
                    <ol><h4>Steps</h4>
 +
                        <li>Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.</li>
 +
                        <li>Inoculate 100 mL of LB broth with 1 mL of the previous culture.</li>
 +
                        <li>Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.</li>
 +
                        <li>Place on ice for 10 minutes.</li>
 +
                        <li>Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.</li>
 +
                        <li>Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.</li>
 +
                        <li>Centrifuge at 4000 rpm for 10 minutes.</li>
 +
                        <li>Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.</li>
 +
                        <li>Prepare 200 μl aliquots and store at -80 °C.</li>
 +
                    </ol>
 +
                </div>
 +
            </div>
 +
        </div>
 +
        <div class = "col-md-2"></div>
 +
    </div>
 +
    <div class="row">
 +
        <div class = "col-md-2"></div>
 +
        <div class="col-md-8">
 +
            <div class="dropdown paragraphU paddingButton">
 +
                <button class="btn btn-primary dropdown-toggle responsiveImage" type="button" data-toggle= "dropdown">Protocol I
 +
                <span class="caret"></span></button>
 +
                <div class="dropdown-menu">
 +
                    <h3>Preparation of chemocompetent cells</h3>
 +
                    <ul><h4>Materials</h4>
 +
                        <li>LB Medium</li>
 +
                        <li>01. M CaCI2</li>
 +
                        <li>CaCL2 0.1M + 15% glycerol</li>
 +
                        <li>Sterile Falcon tubes</li>
 +
                    </ul>
 +
                    <ol><h4>Steps</h4>
 +
                        <li>Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.</li>
 +
                        <li>Inoculate 100 mL of LB broth with 1 mL of the previous culture.</li>
 +
                        <li>Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.</li>
 +
                        <li>Place on ice for 10 minutes.</li>
 +
                        <li>Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.</li>
 +
                        <li>Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.</li>
 +
                        <li>Centrifuge at 4000 rpm for 10 minutes.</li>
 +
                        <li>Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.</li>
 +
                        <li>Prepare 200 μl aliquots and store at -80 °C.</li>
 +
                    </ol>
 +
                </div>
 +
            </div>
 +
        </div>
 +
        <div class = "col-md-2"></div>
 +
    </div>
 +
    <div class="row">
 +
        <div class = "col-md-2"></div>
 +
        <div class="col-md-8">
 +
            <div class="dropdown paragraphU paddingButton">
 +
                <button class="btn btn-primary dropdown-toggle responsiveImage" type="button" data-toggle= "dropdown">Protocol J
 +
                <span class="caret"></span></button>
 +
                <div class="dropdown-menu">
 +
                    <h3>Preparation of chemocompetent cells</h3>
 +
                    <ul><h4>Materials</h4>
 +
                        <li>LB Medium</li>
 +
                        <li>01. M CaCI2</li>
 +
                        <li>CaCL2 0.1M + 15% glycerol</li>
 +
                        <li>Sterile Falcon tubes</li>
 +
                    </ul>
 +
                    <ol><h4>Steps</h4>
 +
                        <li>Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.</li>
 +
                        <li>Inoculate 100 mL of LB broth with 1 mL of the previous culture.</li>
 +
                        <li>Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.</li>
 +
                        <li>Place on ice for 10 minutes.</li>
 +
                        <li>Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.</li>
 +
                        <li>Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.</li>
 +
                        <li>Centrifuge at 4000 rpm for 10 minutes.</li>
 +
                        <li>Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.</li>
 +
                        <li>Prepare 200 μl aliquots and store at -80 °C.</li>
 +
                    </ol>
 +
                </div>
 +
            </div>
 +
        </div>
 +
        <div class = "col-md-2"></div>
 +
    </div>
 +
    <div class="row">
 +
        <div class = "col-md-2"></div>
 +
        <div class="col-md-8">
 +
            <div class="dropdown paragraphU paddingButton">
 +
                <button class="btn btn-primary dropdown-toggle responsiveImage" type="button" data-toggle= "dropdown">Protocol K
 +
                <span class="caret"></span></button>
 +
                <div class="dropdown-menu">
 +
                    <h3>Preparation of chemocompetent cells</h3>
 +
                    <ul><h4>Materials</h4>
 +
                        <li>LB Medium</li>
 +
                        <li>01. M CaCI2</li>
 +
                        <li>CaCL2 0.1M + 15% glycerol</li>
 +
                        <li>Sterile Falcon tubes</li>
 +
                    </ul>
 +
                    <ol><h4>Steps</h4>
 +
                        <li>Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.</li>
 +
                        <li>Inoculate 100 mL of LB broth with 1 mL of the previous culture.</li>
 +
                        <li>Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.</li>
 +
                        <li>Place on ice for 10 minutes.</li>
 +
                        <li>Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.</li>
 +
                        <li>Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.</li>
 +
                        <li>Centrifuge at 4000 rpm for 10 minutes.</li>
 +
                        <li>Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.</li>
 +
                        <li>Prepare 200 μl aliquots and store at -80 °C.</li>
 +
                    </ol>
 +
                </div>
 +
            </div>
 +
        </div>
 +
        <div class = "col-md-2"></div>
 +
    </div>
 +
    <div class="row">
 +
        <div class = "col-md-2"></div>
 +
        <div class="col-md-8">
 +
            <div class="dropdown paragraphU paddingButton">
 +
                <button class="btn btn-primary dropdown-toggle responsiveImage" type="button" data-toggle= "dropdown">Protocol L
 
                 <span class="caret"></span></button>
 
                 <span class="caret"></span></button>
 
                 <div class="dropdown-menu">
 
                 <div class="dropdown-menu">

Revision as of 20:45, 27 October 2017

IGEM_TECCEM

Experiments

Protocols

Preparation of chemocompetent cells

    Materials

  • LB Medium
  • 01. M CaCI2
  • CaCL2 0.1M + 15% glycerol
  • Sterile Falcon tubes

    Steps

  1. Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
  2. Inoculate 100 mL of LB broth with 1 mL of the previous culture.
  3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
  4. Place on ice for 10 minutes.
  5. Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
  6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.
  7. Centrifuge at 4000 rpm for 10 minutes.
  8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.
  9. Prepare 200 μl aliquots and store at -80 °C.

Preparation of chemocompetent cells

    Materials

  • LB Medium
  • 01. M CaCI2
  • CaCL2 0.1M + 15% glycerol
  • Sterile Falcon tubes

    Steps

  1. Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
  2. Inoculate 100 mL of LB broth with 1 mL of the previous culture.
  3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
  4. Place on ice for 10 minutes.
  5. Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
  6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.
  7. Centrifuge at 4000 rpm for 10 minutes.
  8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.
  9. Prepare 200 μl aliquots and store at -80 °C.

Preparation of chemocompetent cells

    Materials

  • LB Medium
  • 01. M CaCI2
  • CaCL2 0.1M + 15% glycerol
  • Sterile Falcon tubes

    Steps

  1. Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
  2. Inoculate 100 mL of LB broth with 1 mL of the previous culture.
  3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
  4. Place on ice for 10 minutes.
  5. Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
  6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.
  7. Centrifuge at 4000 rpm for 10 minutes.
  8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.
  9. Prepare 200 μl aliquots and store at -80 °C.

Preparation of chemocompetent cells

    Materials

  • LB Medium
  • 01. M CaCI2
  • CaCL2 0.1M + 15% glycerol
  • Sterile Falcon tubes

    Steps

  1. Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
  2. Inoculate 100 mL of LB broth with 1 mL of the previous culture.
  3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
  4. Place on ice for 10 minutes.
  5. Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
  6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.
  7. Centrifuge at 4000 rpm for 10 minutes.
  8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.
  9. Prepare 200 μl aliquots and store at -80 °C.

Preparation of chemocompetent cells

    Materials

  • LB Medium
  • 01. M CaCI2
  • CaCL2 0.1M + 15% glycerol
  • Sterile Falcon tubes

    Steps

  1. Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
  2. Inoculate 100 mL of LB broth with 1 mL of the previous culture.
  3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
  4. Place on ice for 10 minutes.
  5. Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
  6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.
  7. Centrifuge at 4000 rpm for 10 minutes.
  8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.
  9. Prepare 200 μl aliquots and store at -80 °C.

Preparation of chemocompetent cells

    Materials

  • LB Medium
  • 01. M CaCI2
  • CaCL2 0.1M + 15% glycerol
  • Sterile Falcon tubes

    Steps

  1. Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
  2. Inoculate 100 mL of LB broth with 1 mL of the previous culture.
  3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
  4. Place on ice for 10 minutes.
  5. Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
  6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.
  7. Centrifuge at 4000 rpm for 10 minutes.
  8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.
  9. Prepare 200 μl aliquots and store at -80 °C.

Preparation of chemocompetent cells

    Materials

  • LB Medium
  • 01. M CaCI2
  • CaCL2 0.1M + 15% glycerol
  • Sterile Falcon tubes

    Steps

  1. Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
  2. Inoculate 100 mL of LB broth with 1 mL of the previous culture.
  3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
  4. Place on ice for 10 minutes.
  5. Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
  6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.
  7. Centrifuge at 4000 rpm for 10 minutes.
  8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.
  9. Prepare 200 μl aliquots and store at -80 °C.

Preparation of chemocompetent cells

    Materials

  • LB Medium
  • 01. M CaCI2
  • CaCL2 0.1M + 15% glycerol
  • Sterile Falcon tubes

    Steps

  1. Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
  2. Inoculate 100 mL of LB broth with 1 mL of the previous culture.
  3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
  4. Place on ice for 10 minutes.
  5. Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
  6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.
  7. Centrifuge at 4000 rpm for 10 minutes.
  8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.
  9. Prepare 200 μl aliquots and store at -80 °C.

Preparation of chemocompetent cells

    Materials

  • LB Medium
  • 01. M CaCI2
  • CaCL2 0.1M + 15% glycerol
  • Sterile Falcon tubes

    Steps

  1. Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
  2. Inoculate 100 mL of LB broth with 1 mL of the previous culture.
  3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
  4. Place on ice for 10 minutes.
  5. Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
  6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.
  7. Centrifuge at 4000 rpm for 10 minutes.
  8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.
  9. Prepare 200 μl aliquots and store at -80 °C.

Preparation of chemocompetent cells

    Materials

  • LB Medium
  • 01. M CaCI2
  • CaCL2 0.1M + 15% glycerol
  • Sterile Falcon tubes

    Steps

  1. Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
  2. Inoculate 100 mL of LB broth with 1 mL of the previous culture.
  3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
  4. Place on ice for 10 minutes.
  5. Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
  6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.
  7. Centrifuge at 4000 rpm for 10 minutes.
  8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.
  9. Prepare 200 μl aliquots and store at -80 °C.

Preparation of chemocompetent cells

    Materials

  • LB Medium
  • 01. M CaCI2
  • CaCL2 0.1M + 15% glycerol
  • Sterile Falcon tubes

    Steps

  1. Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
  2. Inoculate 100 mL of LB broth with 1 mL of the previous culture.
  3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
  4. Place on ice for 10 minutes.
  5. Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
  6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.
  7. Centrifuge at 4000 rpm for 10 minutes.
  8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.
  9. Prepare 200 μl aliquots and store at -80 °C.

Preparation of chemocompetent cells

    Materials

  • LB Medium
  • 01. M CaCI2
  • CaCL2 0.1M + 15% glycerol
  • Sterile Falcon tubes

    Steps

  1. Inoculate an isolated colony of an Escherichia coli strain in a tube containing 10 mL of LB medium and incubate overnight at 37°C and 260 rpm.
  2. Inoculate 100 mL of LB broth with 1 mL of the previous culture.
  3. Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.
  4. Place on ice for 10 minutes.
  5. Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.
  6. Remove the supernatant, resuspend the pellets in 10 mL of cold 0.1 M CaCl2 and allow to incubate on ice for 10 minutes.
  7. Centrifuge at 4000 rpm for 10 minutes.
  8. Remove the supernatant, resuspend the pellet in 2 mL of CaCl2 0.1M + 15% glycerol solution and allow to incubate on ice for 10 min.
  9. Prepare 200 μl aliquots and store at -80 °C.

Experiments

Project Development

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