Difference between revisions of "Team:TecCEM/Experiments"

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                 <div class="dropdown-menu">
 
                 <div class="dropdown-menu">
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                     <h3>Preparation of chemocompetent cells</h3>
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                     <h3>Ligation</h3>
 
                     <ul><h4>Materials</h4>
 
                     <ul><h4>Materials</h4>
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                         <li>LB Medium</li>
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                         <li>Injectable water</li>
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                         <li>01. M CaCI2</li>
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                         <li>T4 DNA ligase buffer (10X)</li>
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                         <li>CaCL2 0.1M + 15% glycerol</li>
+
                         <li>T4 DNA ligase</li>
−
                         <li>Sterile Falcon tubes</li>
+
                         <li>Aligned oligonucleotides</li>
 +
                        <li>Digested linearized plasmid (25ng/μL)</li>
 
                     </ul>
 
                     </ul>
 
                     <ol><h4>Steps</h4>
 
                     <ol><h4>Steps</h4>
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                         <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>Prepare a master mix (if necessary) with the following components:</li>
−
                         <li>Inoculate 100 mL of LB broth with 1 mL of the previous culture.</li>
+
                         <ul>
−
                        <li>Incubate for 3 hours at 37 °C and 260 rpm until it reaches a 0.6 O.D.</li>
+
                            <li>Digested plasmid</li>
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                        <li>Place on ice for 10 minutes.</li>
+
                            <li>Water °MB</li>
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                        <li>Divide the culture in 15 mL Falcon tubes and centrifuge at 5000 rpm for 5 minutes.</li>
+
                            <li>T4 DNA ligase</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>T4 DNA ligase buffer</li>
−
                         <li>Centrifuge at 4000 rpm for 10 minutes.</li>
+
                         </ul>
−
                         <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>Distribute the master mix among the reaction tubes and place the oligonucleotides aligned to have a final volume of 10 μL per reaction.</li>
−
                         <li>Prepare 200 μl aliquots and store at -80 °C.</li>
+
                         <li>Leave incubating at room temperature overnight and transform the next day.</li>                      
 
                     </ol>
 
                     </ol>
 
                 </div>
 
                 </div>

Revision as of 22:43, 27 October 2017

IGEM_TECCEM

Experiments

Protocols

Preparation of chemocompetent cells

    Materials

  • LB Medium
  • CaCl2 0.1 M
  • CaCl2 0.1M + 15% glycerol
  • Desired bacterial strain

    Steps

  1. Inoculate an isolated colony of the desired bacterial strain in a sterile 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 (1:100 proportion).
  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 sterile 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 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 incubate on ice for 10 minutes.
  9. Prepare 200 μl aliquots and store at -80 °C.

Chemocompetent cell transformation

    Materials

  • Chemocompetent cells
  • Plasmid DNA
  • LB or SOC medium

    Previous Steps

  • Prepare a water bath at 42°C.

    Steps

  1. Thaw a tube of competent cells on ice.
  2. Add 5-10 μL of DNA (concentration between 1 pg/mL- 100 ng) to 125μL the competent cells.
  3. Incubate the mixture in ice for 30 minutes.
  4. Place in the water bath at 42°C for 30 seconds and immediately incubate on ice for 5 minutes.
  5. Add 950 μL with SOC or LB medium under sterile conditions.
  6. Incubate at 37°C for 60 minutes at 250 rpm.
  7. Centrifuge at 5000 rpm for 2 minutes to obtain a pellet.
  8. Discard 800 μL from the supernatant and resuspend the pellet in the remaining 200 μL.
  9. Spatulate the volume on LB plates with the selection antibiotic and incubate for 16 hours at 37°C.

Annealing of oligonucleotides

    Materials

  • Oligonucleotides
  • Injectable water
  • NEBuffer 2.1

    Previous Steps

  • Prepare a water bath at 95°C.

    Steps

  1. Perform the corresponding calculations for a desired concentration (μg/μL) and the volume needed to use 5 μg of each oligonucleotide.
  2. For each oligonucleotide, make a mix with 1X NEBuffer 2.1 for a total volume of 20 μl with the following materials:
    • Injectable water
    • NEBuffer 2.1
    • Sense oligonucleotide
    • Antisense oligonucleotide
  3. Incubate the mixture at 95°C for 5 minutes on the water bath.
  4. Leave in the water bath to cool down until room temperature is reached.
  5. Place on ice until further use.

Plasmid digestion

    Materials

  • NEBuffer 2.1
  • Restriction enzymes
  • Injectable water

    Steps

  1. Make a Master Mix with the following:
    • 18 μL of injectable water
    • 5 μL of NEBuffer 2.1
    • 1 μL of Enzyme 1
    • 1 μL of Enzyme 2
  2. Add 6 μL of the Master Mix to 6 μL of linearized plasmid in a sterile tube.
  3. Incubate at 37° for 2 hours.
  4. Inactivate in water bath at 80°C for 20 minutes.
  5. Incubate on ice for immediate use or store at -20 oC.

Ligation

    Materials

  • Injectable water
  • T4 DNA ligase buffer (10X)
  • T4 DNA ligase
  • Aligned oligonucleotides
  • Digested linearized plasmid (25ng/μL)

    Steps

  1. Prepare a master mix (if necessary) with the following components:
    • Digested plasmid
    • Water °MB
    • T4 DNA ligase
    • T4 DNA ligase buffer
  2. Distribute the master mix among the reaction tubes and place the oligonucleotides aligned to have a final volume of 10 μL per reaction.
  3. Leave incubating at room temperature overnight and transform the next day.

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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IGEM_TECCEM