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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Competent Cells

We follow the protocol for the preparation of competent E. coli cells using calcium chloride from Ivaan.com. We use the Competent Cell Test Kit from iGEM to determine our cells' competency.

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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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Glycerol Stocks

  1. Combine 1 mL of 40% glycerol with 1 mL fresh bacterial culture in a cryogenic vial
  2. Gently vortex or pipette to mix
  3. Store culture in -80°C
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Minipreps

We follow the miniprep protocol provided by Thermo Scientific for their GeneJET Plasmid Miniprep Kit. We elute the DNA in 25 µL of Elution Buffer, not 50 µL as stated in the protocol, to achieve a higher concentration.

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Sequencing

We use Genewiz for sequencing and follow their sample submission guidelines.

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Restriction Digests

We use the protocol provided by Thermo Scientific for the fast digestion of DNA as a guide.

  1. For a 20 µL reaction, add:
    • 2 µL enzyme (1 µL each if doing a double digest)
    • 2 µL 10x buffer
    • 1 µg plasmid
    • 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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Agarose Gel Electrophoresis

All gels are 1% agarose unless otherwise stated. We use the 1 kb DNA ladder from NEB.

  1. Dissolve 0.5 g agarose in 50 mL 1X TAE buffer, using the microwave to heat
  2. When cool to the touch, add 5 µL ethidium bromide and pour gel
  3. Run in 1X TAE buffer for 45 min
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Gel Extraction and DNA Cleanup

We follow the protocols provided by Thermo Scientific for their GeneJET Gel Extraction and DNA Cleanup Micro Kit.

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Dephosphorylation

  1. Combine
    • 10 µL DNA
    • 1 µL buffer
    • 0.5 µL rSAP (recombinant shrimp alkaline phosphatase)
  2. Incubate at 37°C for 30 min
  3. Incubate at 65°C for 5 min to deactivate enzymes
  4. Transform 5 uL of each reaction
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Ligation

  1. Combine
    • 1 µL plasmid
    • amount of insert for desired ratio
    • 2 µL buffer
    • Nuclease-free water to 20 µL
    • 1 µL T4 DNA ligase
  2. Incubate at room temperature for 10 min
  3. Incubate at 65°C for 10 min to deactivate enzymes
  4. Transform 5 uL of each reaction
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Polymerase Chain Reaction

We follow the PCR protocol provided by NEB for Phusion High-Fidelity DNA Polymerase.

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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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Annealing Oligos

  1. Combine
    • 1 µg oligos
    • 5 µL 10X T4 DNA Ligase Buffer
    • Nuclease-free water to 50 µL
  2. Incubate at 85°C for 10 min
  3. Cool to 20°C over 30 min
  4. Store annealed oligos at -20°C
Any deviations from this protocol are noted in the Lab Notebook.
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10X TBE Buffer

For 1 liter, combine

  • 108 g Tris base
  • 55 g boric acid
  • 40 mL 0.5 M EDTA (pH = 8.0)
  • water to 1 liter

Buffer B

Combine

  • 25 mM NaCl
  • 50 mM MOPS
  • NaOH to adjust pH to 7.5
  • water to volume

1X PBS

  • For 1 liter, dissolve the following in 800 mL of water
    • 8 g NaCl
    • 0.2 g KCl
    • 1.44 g Na2HPO4
    • 0.24 g KH2PO4
  • Adjust pH to 7.4 using NaOH
  • Add water to volume

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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