Team:Pittsburgh/Protocols

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Office of the Provost Department of Bioengineering Department of Biological Sciences Department of Chemistry Swanson School of Engineering
IDT NEB Experiment

Contents

Pouring Plates

  1. In 1L of DI water, add 25 grams of LB broth and 15 grams of agar. Adjust accordingly if making a different volume
  2. Swirl gently to dissolve some of the LB broth
  3. Autoclave for 20 minutes
  4. Remove from the autoclave and let cool
  5. When cool, add antibiotic to working concentration
    • Ampicillin - 100 µg/mL
    • Chloramphenicol - 25 µg/mL
    • Kanamycin - 50 µg/mL
  6. Pour plates
  7. Let them sit for ~30 min to solidify
  8. Store in cold room in original plate sleeve. Make sure agar side is up
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Transformations

We follow the transformation protocol provided by iGEM. Any deviations from this protocol are noted in the Lab Notebook.


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Liquid Cultures

  1. In a 15 mL centrifuge tube, add 5 mL of LB Broth
  2. Add necessary antibiotic to working concentration
  3. Using a pipette tip, lift colony of interest
  4. Place tip in centrifuge tube
  5. Tape on the lid. Be sure not to twist tightly
  6. Incubate at 37°C in the shaker overnight
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Restriction Digests

  1. For a 20 µL reaction, add:
    1. 2 µL enzyme (1 µL each if doing a double digest)
    2. 2 µL 10x buffer
    3. 1 µg plasmid
    4. Nuclease-free water to volume
  2. Incubate at 37°C for 30 min
  3. Incubate at 65°C for 20 min to deactivate enzymes
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Cell-Free Expression

We follow the protocol provided by NEB for the protein synthesis reaction using PURExpress (E6800).


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50x TAE Buffer

Materials

  • 242 grams Tris free base
  • 18.61 grams Disodium EDTA
  • 57.1 mL glacial acetic acid
  • DI water

Procedure

  1. Add Tris free base and EDTA to ~700 mL of DI water
  2. Stir until dissolved
  3. Autoclave for 20 minutes
  4. Add acetic acid
  5. Adjust the volume with DI water until the volume is 1 L
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