Haynes:Protocols

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** [[Haynes:Assembly101 | Model Procedure for Assembling BioBrick Parts: Classic Ligation for Beginners]]
** [[Haynes:Assembly101 | Model Procedure for Assembling BioBrick Parts: Classic Ligation for Beginners]]
* [[Haynes:Gibson_Assembly | Gibson Assembly]]
* [[Haynes:Gibson_Assembly | Gibson Assembly]]
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* [[Haynes:TypeIIS_Assembly | Type IIS Assembly]] - similar to Golden Gate, Golden Braid, and Moclo
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* [[Haynes:MamCultureMedia | Media formulas]] - cell line-specific formulas
* [[Haynes:MamCultureMedia | Media formulas]] - cell line-specific formulas
* [[Haynes:TransfectionPlasmid_Lipo | Transfection - Lipofectamine]] - Transfection of plasmid DNA into cells with Lipofectamine
* [[Haynes:TransfectionPlasmid_Lipo | Transfection - Lipofectamine]] - Transfection of plasmid DNA into cells with Lipofectamine
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==Protein==
==Protein==
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==Real Time Quantitative PCR==
==Real Time Quantitative PCR==
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* [[Haynes:UPLassay | Universal Probe Library (UPL) assay]]: for the Roche Light Cycler 480
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* [[Haynes:UPLassay | Universal Probe Library (UPL) assay]] - for the Roche Light Cycler 480
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==RNA & cDNA protocols==
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* [[Haynes:TRIzol_RNeasy | RNA mini prep - TRIzol/ RNeasy column]]
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==Resources at OpenWetWare==
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==Other Resources - OpenWetWare==
* [http://openwetware.org/wiki/E._coli_genotypes E. coli Strains]  
* [http://openwetware.org/wiki/E._coli_genotypes E. coli Strains]  
* [http://openwetware.org/wiki/Materials Materials]: buffer formulas; specific information on commonly used reagents (''e.g.'', LB broth, ampicillin, restriction enzymes, etc.)
* [http://openwetware.org/wiki/Materials Materials]: buffer formulas; specific information on commonly used reagents (''e.g.'', LB broth, ampicillin, restriction enzymes, etc.)
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Add dH<sub>2</sub>O 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.
Add dH<sub>2</sub>O 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.
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}}
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'''LB (Lennox) Agar plates''' {{hide|
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Volume: 1 L
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* Bacto-agar, 15 g
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* Caesin tryptone, 10 g
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* Yeast extract, 5 g
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* NaCl, 5 g
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* 1 N NaOH, 1 mL
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Dissolve in 1000 mL dH<sub>2</sub>O. 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.
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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.
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.
}}
}}
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'''Oligo Annealing Buffer, 10x''' {{hide|
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Formula: [1M NaCl; 100 mM Tris-HCl pH 7.4]<br>
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Volume: 1000 μL
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* 5M NaCl, 200 μL
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* 1M Tris-HCl, 100 μL
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* dH<sub>2</sub>O, 700 μL
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Keep frozen at -20°C.
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}}
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Revision as of 18:58, 19 March 2013

link=Haynes_Lab
link=http://engineering.asu.edu/

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Contents

Protocols

DNA Assembly


Cell Culture: Bacteria


Cell Culture: Mammalian


Protein


Real Time Quantitative PCR


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.

LB (Lennox) Agar plates

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)

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

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.



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