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:Research | <font face="trebuchet ms" style="color:#000000"> '''Research''' </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 Info''' </font>]]
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[[Haynes:Publications | <font face="trebuchet ms" style="color:#000000"> '''Publications''' </font>]] &nbsp;&nbsp;&nbsp;
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</div>
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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 style="width: 1000px">
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</div><br>
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=Protocols=
=Protocols=
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'''BioBrick Assembly'''
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==DNA Assembly==
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* [[Haynes:BioBrick Method short | BioBrick assembly overview]]
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* [[Haynes:BioBrick Method short | BioBrick Parts Assembly: an Overview]]
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* [[Haynes:Making BioBricks | Making DNA parts for standardized assembly]]
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** [[Haynes:Making BioBricks | Making Standardized BioBrick Parts]]
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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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'''Cell Culture: Bacteria'''
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<br>
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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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* Coming soon
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==Cell Culture: Mammalian==
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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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==Protein==
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* [[Haynes:Bradford | Bradford assay]] - measure protein concentration
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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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 +
 
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==Resources at OpenWetWare==
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* [http://openwetware.org/wiki/E._coli_genotypes E. coli Strains]
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* [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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 +
 
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==Software Guides==
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* [[Haynes:Jmol_Guide | Jmol commands: controlling PDB structure displays]]
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<br>
=Recipes=
=Recipes=
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Click "show" to expand each recipe, and "hide" to, well, hide it.<br><br>
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<div style="width: 350px">
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'''Bromo-Blue/X-cyanol Loading Buffer, 20X''' {{hide|
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Formula: [60% glycerol; 12.5 mg/mL bromophenol blue; 12.5 mg/mL xylene cyanol]<br>
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Volume: 20 mL
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* 100% glycerol, 12 mL
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* Bromophenol blue, 250 mg
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* Xylene cyanol, 250 mg
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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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}}
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'''Chromatin Prep Buffer A''' {{hide|
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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]<br>
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Volume: 100 mL
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* 1 M HEPES pH 7.9, 1 mL
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* 1 M KCl, 1 mL
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* 1 M MgCl<sub>2</sub>, 150 μL
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* Sucrose, 11.6 g
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* 50% Glycerol, 20 mL
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* 1 M Dithiotreitol (DTT), 100 μL
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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.
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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 Broth (Lennox)''' {{hide|
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Volume: 1 L
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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 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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}}
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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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{| class="wikitable" width="300px"
 
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| '''LB Broth (Lennox)''' || &nbsp;
 
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|-
 
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| Caesin tryptone* || 10 g
 
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|-
 
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| Yeast extract* || 5 g
 
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|-
 
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| NaCl* || 5 g
 
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|}
 
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* Or 25 g
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</div>

Revision as of 21:32, 16 November 2012

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

   Home     Research     Publications     People     Contact     Protocols     Lab Resources     Personnel Info

Contents

Protocols

DNA Assembly


Cell Culture: Bacteria


Cell Culture: Mammalian

Protein


Real Time Quantitative PCR


Resources at 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 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.


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