Haynes:Protocols

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Protocols

DNA Assembly

• BioBrick Parts Assembly: an Overview
  • Making Standardized BioBrick Parts
  • Model Procedure for Assembling BioBrick Parts: Classic Ligation for Beginners
• Gibson Assembly

Cell Culture: Bacteria

• Chemically competent cell prep
• Glycerol Stocks - for long term -80°C storage
• Transformation of E. coli with plasmid DNA

Cell Culture: Mammalian

• Media formulas - cell line-specific formulas

Protein

• Bradford assay - measure protein concentration

Real Time Quantitative PCR

• Universal Probe Library (UPL) assay: for the Roche Light Cycler 480

Resources at OpenWetWare

• E. coli Strains
• Materials: buffer formulas; specific information on commonly used reagents (e.g., LB broth, ampicillin, restriction enzymes, etc.)

Software Guides

• Jmol commands: controlling PDB structure displays

Recipes

Click "show" to expand each recipe, and "hide" to, well, hide it.

Bromo-Blue/X-cyanol Loading Buffer, 10X [show][hide]

Formula: [30% glycerol; 12.5 mg/mL bromophenol blue; 12.5 mg/mL xylene cyanol]
Volume: 20 mL

• 50% glycerol, 12 mL
• Bromophenol blue, 250 mg
• Xylene cyanol, 250 mg

Bring up to total volume with dH₂O.

Chromatin Prep Buffer A [show][hide]

Formula: [10 mM HEPES (pH 7.9); 10 mM KCl, 1.5 mM MgCl2; 0.34 M sucrose; 10% glycerol; 1 mM dithiothreitol; 1x protease inhibitor cocktail]
Volume: 100 mL

• 1 M HEPES pH 7.9, 1 mL
• 1 M KCl, 1 mL
• 1 M MgCl₂, 150 μL
• Sucrose, 11.6 g
• 50% Glycerol, 20 mL
• 1 M Dithiotreitol (DTT), 100 μL

Bring up to total volume with dH₂O. Store at 4°C. Add 100x protease inhibitor cocktail* (1:100) immediately before use.

LB Broth (Lennox) [show][hide]

Volume: 1 L

• Caesin tryptone, 10 g
• Yeast extract, 5 g
• NaCl, 5 g
• 1 N NaOH, 1 mL

Dissolve in 1000 mL dH₂O. Autoclave to sterilize. Cool to room temperature before adding antibiotics. Shortcut: Use 25 g Acros LB Broth, Lennox (BP9722-500) granules instead of caesin tryptone, yeast extract, and NaCl.

Mammalian Cell Media [show][hide]

Volume: 500 mL

• Appropriate incomplete medium, 500 mL
• Fetal bovine serum (FBS), 50 mL
• Penicillin/ streptomycin (pen-strep), 5 mL
• Appropriate antibiotics, depending upon formula

Add FBS and pen-strep directly to the incomplete medium in the original stock bottle. Cap and invert to mix. Filter sterilize using a vacuum filtration unit.