Team:TecCEM/experiments

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.

Experiments

Project Development

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean maximus, odio eu ornare bibendum, tellus lorem mattis ante, non iaculis leo lorem id justo. In placerat sapien eget ultrices venenatis. Vivamus velit augue, efficitur sit amet commodo a, tristique at purus. Aenean quam mi, mollis ac posuere id, faucibus quis velit. Nullam interdum enim nec ultrices volutpat. Proin vel mi eget lorem laoreet venenatis. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Fusce sodales porta mauris, sit amet elementum eros aliquet eu. Pellentesque sed sapien at enim sollicitudin sollicitudin vitae vel diam. Vivamus placerat aliquet enim.

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean maximus, odio eu ornare bibendum, tellus lorem mattis ante, non iaculis leo lorem id justo. In placerat sapien eget ultrices venenatis. Vivamus velit augue, efficitur sit amet commodo a, tristique at purus. Aenean quam mi, mollis ac posuere id, faucibus quis velit. Nullam interdum enim nec ultrices volutpat. Proin vel mi eget lorem laoreet venenatis. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Fusce sodales porta mauris, sit amet elementum eros aliquet eu. Pellentesque sed sapien at enim sollicitudin sollicitudin vitae vel diam. Vivamus placerat aliquet enim.

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean maximus, odio eu ornare bibendum, tellus lorem mattis ante, non iaculis leo lorem id justo. In placerat sapien eget ultrices venenatis. Vivamus velit augue, efficitur sit amet commodo a, tristique at purus. Aenean quam mi, mollis ac posuere id, faucibus quis velit. Nullam interdum enim nec ultrices volutpat. Proin vel mi eget lorem laoreet venenatis. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Fusce sodales porta mauris, sit amet elementum eros aliquet eu. Pellentesque sed sapien at enim sollicitudin sollicitudin vitae vel diam. Vivamus placerat aliquet enim.

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean maximus, odio eu ornare bibendum, tellus lorem mattis ante, non iaculis leo lorem id justo. In placerat sapien eget ultrices venenatis. Vivamus velit augue, efficitur sit amet commodo a, tristique at purus. Aenean quam mi, mollis ac posuere id, faucibus quis velit. Nullam interdum enim nec ultrices volutpat. Proin vel mi eget lorem laoreet venenatis. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Fusce sodales porta mauris, sit amet elementum eros aliquet eu. Pellentesque sed sapien at enim sollicitudin sollicitudin vitae vel diam. Vivamus placerat aliquet enim.

IGEM_TECCEM