Wittrup:Buffers and media

Buffers

• Wittrup: EDTA
• Wittrup: TE (pH 8.0)
• Wittrup: PBS (pH 7.4)
• Wittrup: HBS (pH 7.4)
• Wittrup: TAE (Tris-Acetate)
• Wittrup: TBE (Tris-Borate)

Yeast Media

Introduction

• YPD (complex): No selection, rich medium, very good cell density
• SD-CAA, SG-CAA (Minimal): Trp, Ura, Ade selection, average cell density

Complex Media

• YPD, 1000 mL
  • 10g Yeast extract
  • 20g Peptone
  • 20g Dextrose (D-glucose)

Add ddH2O to final volume. Autoclave or filter sterilize. The dextrose may carmelize in the autoclave, which shouldn't hurt the media, but will make it dark. For filter sterilization, it will be very thick, so use a vacu-cap Gelman filter.

• Agar Plates, 1000 mL
  • 15g Bacto-agar, in addition to above ingredients

Add ddH2O to final volume. Autoclave.

Minimal Media

1000 mL, liquid

• Carbon source
  • SD: 20g Dextrose (D-glucose)
  • SG: 20g Galactose
• Nitrogen source
  • 6.7g Yeast N2 base
• Amino acid source
  • 5.0g CAA (-ADE, -URA, -TRP)
  • Or, Synthetic CAA..
• Buffer
  • pH 6.0 phosphate buffer (standard)
    • 10.19g Na2HPO4.7H2O
    • 8.56g NaH2PO4.H2O
  • pH 4.5 citrate buffer (optional: lower pH discourages bacterial growth)
    • 14.70g sodium citrate
    • 4.29g citric acid monohydrate

Add ddH2O to final volume. Filter sterilize.

1000 mL, Plates

• Filter Sterilize:
  • Carbon source, Nitrogen source, Amino acid source in 0.1*(Final Volume)
• Autoclave:
  • Buffer salts, 15-18g agar, 182g sorbitol in 0.9*(Final Volume)
• Add first mixture to the autoclaved mixture after it has cooled to below 50 oC (touchable by bare hand)

LB/Amp Plates

SOC Broth