Biomolecular Breadboards:Protocols:Crude Extract Prep

Protocol for E. Coli Crude Extract Preparation, 3X

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

From Shin J, Noireaux V: Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70. Journal of Biological Engineering 2010, 4:8

http://www.jbioleng.org/content/4/1/8