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
• Ligase Cycling Reaction - ligation of blunt-ended fragments guided by overlapping oligo "bridges"

Bioinformatics

• ChIP in GALAXY - How to use GALAXY and a large BED file from your hard drive to create a track for the UCSC browser. Useful for data that is too large to create a custom track directly on the UCSC website.
• ChIP on Promoters - How to find chromatin protein enrichment at gene promoters of interest for small sets of genes or all 23,000 human genes, using public ChIP data
• Gene Ontology Term Enrichment - How to compute which standardized terms (GO terms) for cell behavior/ biology are significantly over-represented in a user-defined list of genes

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
• Thawing Cells - starting a new culture from a -150°C frozen stock vial
• Advancing Cells - transferring a new thawed culture into a larger growth vessel
• Splitting Cells - splitting or passaging cells
• Transfection - Lipofectamine - Transfection of plasmid DNA into cells with Lipofectamine
• Cryopreserving Cells - Preparing frozen cell stocks

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 extracted protein concentration
• ELISA assay - specific antibody-based quantification of protein; simpler than Western, but no protein size data
• Fluorescent Protein - measure extracted fluorescent protein levels
• Luciferase assay - measuring firefly luciferase reporter activity with D-luciferin substrate
• Protein Extraction - extracting proteins from mammalian cells
• PAGE - polyacrylamide gel electrophoresis (PAGE)
• Western Blot - gel-to-membrane protein transfer; detection with antibodies

Real Time Quantitative PCR

• Roche Universal Probe Library (UPL) assay - hydrolysis probe PCR for the Roche Light Cycler 480
• Roche Data Analysis - Tips for doing data analysis (from a user's perspective)
• SYBR Green assay - dsDNA-binding fluorescent dye-based detection

RNA & cDNA protocols

• RNA mini prep - TRIzol/ RNeasy column
• cDNA Synthesis - procedures and reaction table templates

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

Detailed protocol

[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)

Detailed protocol

[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.

Resuspension Buffer - for plasmid minipreps [show][hide]

Formula: [25 mM Tris-HCl, 10 mM EDTA, 100 μg/mL RNaseA, pH 8.0] (based on the Sigma formulation)
Volume: 50 mL

• 1M Tris-HCl pH ~8, 2.0 mL
• 500 mM EDTA, 1.0 mL
• Molecular biology grade dH₂O, up to 45 mL
  • Adjust pH to 8.0
• Molecular biology grade dH₂O, up to 50 mL
• 20 mg/mL RNaseA, 250 μL

In a 50 mL conical, add Tris-HCl and EDTA. Fill up to ~45 mL with molecular biology grade dH₂O. Adjust pH to 8.0: add 10 μL acid or base, cap tightly, invert to mix, then measure pH. Repeat as needed. Fill up to 50 mL with molecular biology grade dH₂O. Cap tightly and invert to mix. Keep refrigerated at 4°C.

Tris-acetate-EDTA (TAE) Buffer, 50x [show][hide]

Formula: [2 M Tris, 1 M acetate, 50 mM EDTA]
Volume: 500 mL

• Tris base (FW 121.14), 121 g
• Glacial acetic acid, 28.55 mL
• 500 mM EDTA, 50 mL

Dissolve Tris base in 200 mL dH₂O (in a beaker with stirring). Add (by pipetting) glacial acetic acid and EDTA. Transfer solution to a graduated cylinder and fill up to 500 mL with dH₂O. Transfer to a screw-cap bottle, loosen the cap and secure it to the bottle with autoclave tape. Autoclave in a pan filled about 2 in. deep with water (to prevent boiling-over) to sterilize.

Tris-glycine concentrate [show][hide]

Formula: [250 mM Tris; 1.92 M glycine; pH approx. 8.3]
Volume: 500 mL

• Tris base (FW 121.14), 15.15 g
• Glycine, 72.0 g

Dissolve Tris base and glycine in 200 mL dH₂O (in a beaker with stirring). Transfer solution to a graduated cylinder and fill up to 500 mL with dH₂O. Do not autoclave.