Protocols

Growth Media:

LB (Luria Bertani) medium
  • 1% tryptone
  • 0.5% yeast extract
  • 0.5% sodium chloride
  • 0.1% glucose
  • Para meio seletivo, adicione ampicilina a uma concentração final de 200 μg/mL
YPD (yeast extract + peptone + dextrose) medium
  • 1% yeast extract
  • 2% peptone
  • 2% glucose
Minimal medium
  • 0.67% YNB (Yeast Nitrogen Base) with ammonium sulfate
  • 2% glucose
Sporulation medium
  • 0.5% yeast extract
  • 1% potassium acetate
  • 0.05% glucose

For solid growth media, add 2% bacteriological agar For minimal and sporulation media, add the amino acids and nitrogen bases as needed at the following concentrations:

  • Adenina, uracila: 2 mg/mL
  • Leucina, histidina, triptofano: 10 mg/mL

Preparation of competent E. coli cells:

  1. Inoculate a loop of cells from a reasonably fresh plate into 50 mL of LB.
  2. Shake at 37ºC until a Klett of 100 is reached. With experience, this can be estimated visually. It may take about 4 hours.
  3. Transfer the cells to a sterile 50 mL plastic tube.
  4. Centrifuge at 3,500 rpm for 5 minutes at 4ºC.
  5. Resuspend the cells in 10 mL of sterile 10 mM calcium chloride by gently drawing up in a 10 mL pipette.
  6. Centrifuge again at 3,500 rpm for 5 minutes at 4ºC.
  7. Resuspend the cells in 10 mL of sterile 30 mM calcium chloride, with 10 mM Tris (pH 7.5). Let sit on ice for at least 30 minutes.
  8. Centrifuge as described above.
  9. Resuspend the cells in 5 mL of sterile 30 mM calcium chloride, with 10 mM Tris (pH 7.5).
  10. The cells are now competent and ready to be used for transformations. They can be stored overnight on ice or frozen in the presence of 7% DMSO.

    11. Transformation of competent E. coli cells:

    1. Transfer 0.2 mL of competent cells to a sterile 1.5 mL Eppendorf tube.
    2. Add DNA. Incubate on ice for 10 minutes.
    3. Heat shock cells by incubating for 2 minutes at 42ºC. Incubate on ice for 45 seconds.
    4. Add 1 mL of fresh LB. Shake at 37ºC for 30 minutes to 1 hour.

    Yeast transformation by LiAc method:

    1. Incubate cells into 10 mL YPD and let them grow overnight shaking at 30ºC.
    2. Inoculate from overnight into fresh 10 mL YPD so that OD at 600 nm is approximately 0.1. 500 μL from overnight may be enough.
    3. Grow cells shaking at 30ºC until OD at 600 nm reaches 0.6 to 1.0. Centrifuge at 1.5 K for 3 minutes at room temperature.
    4. Resuspend cells in 5 mL TEL (10 mM Tris, pH 7.5, 1 mM EDTA, 0.1 M lithium acetate). Centrifuge at 1.5 K for 3 minutes at room temperature.
    5. Resuspend cells in a minimum of 300 μL TEL. Transfer 100 μL to a sterile 1.5 mL Eppendorf tube.
    6. Add a droplet of 5 μL of 10 mg/mL carrier salmon sperm DNA to the tube wall. Over this droplet, add 5 μL of transforming DNA (1-10 μg). Mix the DNA droplet with the cells by pipetting and incubate for 15 minutes at room temperature without shaking.
    7. Add 400 μL of 40% polyethylene glycol (4,000) in TEL buffer and mix by pipetting. Incubate for 40 minutes at room temperature without shaking.
    8. Heat shock cells by incubating for 10 minutes at 42ºC.
    9. Add 700 μL of TE (10 mM Tris, pH 7.5, 1 mM EDTA).
    10. Centrifuge in microfuge for 30 seconds at room temperature. Remove supernatant. Resuspend cells in 200 μL of TE. Centrifuge in microfuge for 30 seconds at room temperature. Once again, resuspend cells in 200 μL of TE.
    11. Spread on selective medium (minimal medium with selectively added amino acids and nitrogen bases).
    12. Incubate at 30ºC. Cell colonies may be observed after 2-3 days.

      13. Modified plasmid miniprep:

      1. Add 100 μL of lysis buffer to 1.5 mL Eppendorf tubes (as much as needed for the number of colonies to be screened). Maintain them on ice.
      2. Collect cells from plate with toothpick and transfer into the 1.5 mL Eppendorf tube prepared with lysis buffer. Maintain on ice for 1 minute.
      3. Add 200 μL of a mixture of 0.2 M NaOH plus 1% SDS. Maintain cells on ice.
      4. Rub toothpick against the tube wall so that all the cells are suspended in the lysis buffer. Discard the toothpick and maintain cells on ice.
      5. Add 150 μL of 7.5 M ammonium acetate (57.8 g ammonium acetate, 2 mL of glacial acetic acid in 100 mL). Mix by inversion 3 times and centrifuge in microfuge for 3 minutes. Transfer supernatant (where the plasmid DNA is) to a new 1.5 mL Eppendorf tube.
      6. Mix by inversion 5 times and centrifuge in microfuge for 5 minutes. Note that DNA should precipitate at this step, although the small pellet may not be seen by the naked eye.
      7. Discard supernatant and wash twice with 80% ethanol.
      8. Dry in speedvac. Dissolve pellet in water (5 μL as a suggestion).
      Lysis buffer:
      • 1 mL 25 mM Tris, pH 8.0, 125 mM EDTA
      • 1 mL 0.25 M sucrose
      • 20 μL 10 mg/mL ribonuclease
      • 5 mg lysozyme

      Preparation of plasmid DNA by Triton X-100 lysis procedure:

      1. Scrape cells with sterile spatula from LB+ampicillin plate and suspend in 2 mL lysozyme mix in a 15 mL plastic tube. Incubate for 30 minutes on ice.
      2. Add 1 mL of Triton X-100 lysis mix. Mix by inversion transfer to thick walled Beckmann tube and centrifuge at 65 K for 15 minutes at 4ºC.
      3. Transfer supernatant (where the plasmid DNA is) to a 15 mL plastic tube and extract with equal volume of water-saturated phenol. Centrifuge at 4 K for 5 minutes. Collect upper phase (aqueous phase).
      4. Add a mixture of chloroform:isoamyl alcohol (24:1) with equal volume of the previously collected aqueous phase. Centrifuge at 4 K for 5 minutes. Collect upper phase.
      5. Add 1/20th volumes of 5 M sodium chloride and 3 volumes of ethanol. Centrifuge at 4 K for 10 minutes. Note that DNA should precipitate as an oily pellet.
      6. Discard supernatant. Dissolve the pellet in 1.5 mL of 2 M ammonium acetate and precipitate with 4.5 mL of ethanol. Note that DNA should precipitate as a whitish pellet. Wash twice with 80% ethanol.
      7. Dry in speedvac. Dissolve pellet in 100 μL water.
      Lysozyme mix:
      • 1 mL 25 mM Tris, pH 8.0, 125 mM EDTA
      • 1 mL 0.25 M sucrose
      • 20 μL 10 mg/mL ribonuclease
      • 5 mg lysozyme
      Lysis mix:
      • 45 mL water
      • 37 mL 0.5 M EDTA
      • 15 mL 1 M Tris, pH 8.0
      • 3 mL 10% Triton X-100