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
• Type IIS Assembly - similar to Golden Gate, Golden Braid, and Moclo

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
• Transfection - Lipofectamine - Transfection of plasmid DNA into cells with Lipofectamine
• Splitting Cells - passaging cells
• Thawing Cells - starting a new culture from a -150°C frozen stock vial

Imaging: Mammalian

Flow Cytometry

• Detecting Fluorescent Protein Expression
• Intracellular Protein Staining with Saponin Permeabilization - how to detect cytoplasmic proteins using antibodies

Microscopy

• Immunocytology - staining fixed cells with antibodies

Protein & Enzyme Assays

• Bradford assay - measure protein concentration
• ELISA assay - specific antibody-based quantification of protein; simpler than Western, but no protein size data
• Luciferase assay - Measuring firefly luciferase reporter activity with D-luciferin substrate

Real Time Quantitative PCR

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

RNA & cDNA protocols

• RNA mini prep - TRIzol/ RNeasy column

Other Resources - 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, 20X [show][hide]

Formula: [60% glycerol; 12.5 mg/mL bromophenol blue; 12.5 mg/mL xylene cyanol]
Volume: 20 mL

• 100% glycerol, 12 mL
• Bromophenol blue, 250 mg
• Xylene cyanol, 250 mg

Start with 18 mL dH₂O. Add in glycerol 6 mL at a time and mix by pipetting up and down several times (it is very viscous). Add dyes.

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.

DNA Ladder mix, Gene Ruler 1 kb Plus [show][hide]

Formula: [0.5 ng/ 10 μL Fermentas Gene Ruler 1 kb Plus; 1x loading dye]
Volume: 1000 μL

• Gene Ruler plus (0.5 ng/μL), 100 μL
• 20x loading dye, 50 μL
• dH₂O, 850 μL

Add dH₂O directly to the supplier's tube (containing 100 μL of ladder). Add the 20x loading dye. Mix. Aliquot 200 μL portions into five 1.5 mL tubes. Store at -20°C.

LB (Lennox) Agar plates [show][hide]

Volume: 1 L

• Bacto-agar, 15 g
• 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 ~50°C (warm to touch) before adding antibiotics. Add antibiotic(s) and pour into sterile petri dishes. 1L should yield about 40 plates. Shortcut: Use 25 g Acros LB Broth, Lennox (BP9722-500) granules instead of caesin tryptone, yeast extract, and NaCl.

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.

Oligo Annealing Buffer, 10x [show][hide]

Formula: [1M NaCl; 100 mM Tris-HCl pH 7.4]
Volume: 1000 μL

• 5M NaCl, 200 μL
• 1M Tris-HCl, 100 μL
• dH₂O, 700 μL

Keep frozen at -20°C.