Haynes:Protocols
Protocols
DNA Assembly
- BioBrick Parts Assembly: an Overview
- Gibson Assembly
- Type IIS Assembly - similar to Golden Gate, Golden Braid, and Moclo
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
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
- Roche Universal Probe Library (UPL) assay - for the Roche Light Cycler 480
- Roche Data Analysis - Tips for doing data analysis (from a user's perspective)
RNA & cDNA protocols
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
Recipes
Click "show" to expand each recipe, and "hide" to, well, hide it.
Bromo-Blue/X-cyanol Loading Buffer, 20X
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
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
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.
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.
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
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
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.