Haynes:Protocols

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[[Image:Hayneslab3.gif|link=Haynes_Lab]] [[Image:Asu_logo_3.gif|right|link=http://engineering.asu.edu/]]<br>
[[Image:Hayneslab3.gif|link=Haynes_Lab]] [[Image:Asu_logo_3.gif|right|link=http://engineering.asu.edu/]]<br>
&nbsp;&nbsp;[[Haynes_Lab | <font face="trebuchet ms" style="color:#000000"> '''Home''' </font>]] &nbsp;&nbsp;&nbsp;
&nbsp;&nbsp;[[Haynes_Lab | <font face="trebuchet ms" style="color:#000000"> '''Home''' </font>]] &nbsp;&nbsp;&nbsp;
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[[Haynes:Publications | <font face="trebuchet ms" style="color:#000000"> '''Publications''' </font>]] &nbsp;&nbsp;&nbsp;
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[[Haynes:Lab Members | <font face="trebuchet ms" style="color:#000000"> '''People''' </font>]] &nbsp;&nbsp;&nbsp;
[[Haynes:Contact | <font face="trebuchet ms" style="color:#000000"> '''Contact''' </font>]] &nbsp;&nbsp;&nbsp;
[[Haynes:Contact | <font face="trebuchet ms" style="color:#000000"> '''Contact''' </font>]] &nbsp;&nbsp;&nbsp;
[[Haynes:Protocols | <font face="trebuchet ms" style="color:#000000"> '''Protocols''' </font>]] &nbsp;&nbsp;&nbsp;
[[Haynes:Protocols | <font face="trebuchet ms" style="color:#000000"> '''Protocols''' </font>]] &nbsp;&nbsp;&nbsp;
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[[Haynes:TechResources | <font face="trebuchet ms" style="color:#000000"> '''Technical Resources''' </font>]] &nbsp;&nbsp;&nbsp;
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[[Haynes:TechResources | <font face="trebuchet ms" style="color:#000000"> '''Lab Resources''' </font>]] &nbsp;&nbsp;&nbsp;
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[[Haynes:Lab Members | <font face="trebuchet ms" style="color:#000000"> '''People''' </font>]] &nbsp;&nbsp;&nbsp;
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[[Haynes:Internal | <font face="trebuchet ms" style="color:#000000"> '''Personnel''' </font>]] &nbsp;&nbsp;&nbsp;
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[[Haynes:Publications | <font face="trebuchet ms" style="color:#000000"> '''Publications''' </font>]] &nbsp;&nbsp;&nbsp;
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[[Haynes:Calendar | <font face="trebuchet ms" style="color:#000000"> '''Calendar''' </font>]]
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[[Haynes:Research | <font face="trebuchet ms" style="color:#000000"> '''Research''' </font>]] &nbsp;&nbsp;&nbsp;
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[[Haynes:Internal | <font face="trebuchet ms" style="color:#000000"> '''Internal Info''' </font>]]  
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</div><br>
</div><br>
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<div style="width: 1000px">
=Protocols=
=Protocols=
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'''BioBrick Assembly'''
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==DNA Assembly==
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* [[Haynes:BioBrick Method short | Assembly of BioBrick Parts: an Overview]]
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* [[Haynes:BioBrick Method short | BioBrick Parts Assembly: an Overview]]
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* [[Haynes:Making BioBricks | Making Standardized DNA Parts]]
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** [[Haynes:Making BioBricks | Making Standardized BioBrick Parts]]
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* [[Haynes:Assembly101 | Model Procedure for Assembling Parts: Classic Ligation for Beginners]]
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** [[Haynes:Assembly101 | Model Procedure for Assembling BioBrick Parts: Classic Ligation for Beginners]]
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* [[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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<br>
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'''Cell Culture: Bacteria'''
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==Cell Culture: Bacteria==
* [[Haynes:ChemComp cells | Chemically competent cell prep]]
* [[Haynes:ChemComp cells | Chemically competent cell prep]]
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* [[Haynes:Glycerol Stocks | Glycerol Stocks]] - for long term -80°C storage
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* [[Haynes:TransformationPlasmids | Transformation of E. coli with plasmid DNA]]
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'''Cell Culture: Mammalian'''
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==Cell Culture: Mammalian==
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* Coming soon
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* [[Haynes:MamCultureMedia | Media formulas]] - cell line-specific formulas
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* [[Haynes:TransfectionPlasmid_Lipo | Transfection - Lipofectamine]] - Transfection of plasmid DNA into cells with Lipofectamine
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* [[Haynes:SplittingCells | Splitting Cells]] - passaging cells
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* [[Haynes:ThawingCells | Thawing Cells]] - starting a new culture from a -150°C frozen stock vial
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'''Resources at OpenWetWare'''
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==Imaging: Mammalian==
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'''Flow Cytometry'''
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* [[Haynes:FCMamalian_FluorGene | Detecting Fluorescent Protein Expression]]
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* [[Haynes:ICCSaponin | Intracellular Protein Staining with Saponin Permeabilization]] - how to detect cytoplasmic proteins using antibodies
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'''Microscopy'''
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* [[Haynes:Mamalian_IFC | Immunocytology]] - staining fixed cells  with antibodies
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==Protein & Enzyme Assays==
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* [[Haynes:Bradford | Bradford assay]] - measure protein concentration
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* [[Haynes:ELISA | ELISA assay]] - specific antibody-based quantification of protein; simpler than Western, but no protein size data
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* [[Haynes:Luciferase | Luciferase assay]] - Measuring firefly luciferase reporter activity with D-luciferin substrate
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 +
==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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==RNA & cDNA protocols==
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* [[Haynes:TRIzol_RNeasy | RNA mini prep - TRIzol/ RNeasy column]]
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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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==Software Guides==
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* [[Haynes:Jmol_Guide | Jmol commands: controlling PDB structure displays]]
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<div style="width: 350px">
<div style="width: 350px">
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'''Bromo-Blue/X-cyanol Loading Buffer, 10X''' {{hide|  
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'''Bromo-Blue/X-cyanol Loading Buffer, 20X''' {{hide|  
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Formula: [30% glycerol; 12.5 mg/mL bromophenol blue; 12.5 mg/mL xylene cyanol]<br>
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Formula: [60% glycerol; 12.5 mg/mL bromophenol blue; 12.5 mg/mL xylene cyanol]<br>
Volume: 20 mL
Volume: 20 mL
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* 50% glycerol, 12 mL
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* 100% glycerol, 12 mL
* Bromophenol blue, 250 mg
* Bromophenol blue, 250 mg
* Xylene cyanol, 250 mg
* Xylene cyanol, 250 mg
-
Bring up to total volume with dH<sub>2</sub>O.
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Start with 18 mL dH<sub>2</sub>O. Add in glycerol 6 mL at a time and mix by pipetting up and down several times (it is very viscous). Add dyes.
}}
}}
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Bring up to total volume with dH<sub>2</sub>O. Store at 4°C. Add 100x protease inhibitor cocktail* (1:100) immediately before use.
Bring up to total volume with dH<sub>2</sub>O. Store at 4°C. Add 100x protease inhibitor cocktail* (1:100) immediately before use.
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}}
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'''DNA Ladder mix, Gene Ruler 1 kb Plus''' {{hide|
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Formula: [0.5 ng/ 10 μL Fermentas Gene Ruler 1 kb Plus; 1x loading dye]<br>
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Volume: 1000 μL
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* Gene Ruler plus (0.5 ng/μL), 100 μL
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* 20x loading dye, 50 μL
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* dH<sub>2</sub>O, 850 μL
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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.
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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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Dissolve in 1000 mL dH<sub>2</sub>O. 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.
Dissolve in 1000 mL dH<sub>2</sub>O. 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.
}}
}}
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'''Mammalian Cell Media''' {{hide|
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Volume: 500 mL
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* [[Haynes:MamCultureMedia | Appropriate incomplete medium]], 500 mL
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* Fetal bovine serum (FBS), 50 mL
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* Penicillin/ streptomycin (pen-strep), 5 mL
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* [[Haynes:MamCultureMedia | Appropriate antibiotics]], depending upon formula
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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.
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}}
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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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</div>
</div>

Revision as of 11:08, 25 August 2013

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

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Contents

Protocols

DNA Assembly


Cell Culture: Bacteria


Cell Culture: Mammalian


Imaging: Mammalian

Flow Cytometry

Microscopy

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


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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