Biomolecular Breadboards:Protocols:Crude Extract Prep

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Protocol for E. Coli 3x Crude Extract Preparation

Time needed: 3 days

Materials

2xYT:

  • 2xYT-broth powder (MP biomedicals)
  • Cat #: 3012-032

Bacto-Agar:

  • Bacto-agar (BD)
  • Product number: 214010

Bead:

  • 0.1mm diameter glass beads (Biospec products)
  • Catalog #: 11079101

Bead beater:

  • Mini-Beadbeater-1 (Biospec products)
  • Catalog #: 3110BX

Bead beating tube:

  • Polypropylene microvials (Biospec products)
  • Catalog #: 522S

Bead filter:

  • Micro bio-spin chromatography columns (Bio-Rad)
  • Product number: 732-6204

Centrifuge (big size):

  • Allegra 64R high-speed refrigerated benchtop centrifuge (Beckman Coulter)

Centrifuge (ultra size):

  • Beckman J2-M1 (Beckman Coulter)

Centrifuge bottle (50ml):

  • Polyallomer bottle with screw-on cap (Beckman Coulter)
  • Part number: 357003

Centrifuge bottle (500ml):

  • Centrifuge bottle assembly polycarbonate (Beckman Coulter)
  • Part number: 355607

Chloramphenicol:

  • Chloramphenicol powder (Sigma-Aldrich)
  • Product number: C1919

Cuvette:

  • Fisherbrand Disposable Cuvettes
  • Cat#: 14-955-127

Dialysis cassette:

  • Slide-A-Lyzer Dialysis Cassette, 10MWCO (Thermo Scientific), maximum capacity: 3mL
  • Product number: 66380

E. coli strain:

  • BL21 Rosetta 2 competent cell (Novagen)
  • Product number: 71402

Electrode:

  • Electrode, 3-in-1 for Φ 400, 500 series pH meter, 12x 150 mm, Epoxy
  • Product number: A57198

Erlenmeyer glass flask:

  • KIMAX erlenmeyer glass flask
  • Product number: 26500 (4000ml)

Floater:

  • Float Buoys: comes with Slide-A-Lyzer Dialysis cassette

Incubator shaker:

  • Incubator shaker (New Brunswick)
  • Innova 4300

K-glutamate:

  • L-Glutamic acid potassium salt monohydrate (Sigma Aldrich)
  • Product number: G1149

Liquid nitrogen

Magnetic stirrer:

  • Thermolyne Nuova II
  • Fisher Scienetific Isotemp Digital Magnetic Stirrer

Mg-glutamate:

  • L-Glutamic acid Hemimagnesium salt tetrahydrate (Sigma Aldrich)
  • Product number: 49605

PH meter:

  • pH meter, Φ 410 pH/mV meter, waterproof, handheld
  • Product number: A58734

Potassium phosphate monobasic solution:

  • Potassium phosphate monobasic solution (Sigma Aldrich)
  • Product number: P8709

Potassium phosphate dibasic solution:

  • Potassium phosphate dibasic solution (Sigma Aldrich)
  • Product number: P8584

Protein assay:

  • Bio-rad Protein Assay Dye Reagent concentrate (Bio-Rad)
  • Product number: 500-0006

Spectrometer:

  • Genesys 10 uv (Thermo Electron corporation)

Table top centrifuge:

  • Microcentrifuge model 5415D (Eppendorf)

Table top vortex:

  • Analog vortex mixer (VWR)
  • Catalog number: 58816-121

Tris:

  • Tris base, white crystals or crystalline powder, molecular biology (Fisher Scientific)
  • Catalog number: BP 1 52 1
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