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;
 
[[Haynes:Publications | <font face="trebuchet ms" style="color:#000000"> '''Publications''' </font>]] &nbsp;&nbsp;&nbsp;
[[Haynes:Publications | <font face="trebuchet ms" style="color:#000000"> '''Publications''' </font>]] &nbsp;&nbsp;&nbsp;
[[Haynes:Lab Members | <font face="trebuchet ms" style="color:#000000"> '''People''' </font>]] &nbsp;&nbsp;&nbsp;
[[Haynes:Lab Members | <font face="trebuchet ms" style="color:#000000"> '''People''' </font>]] &nbsp;&nbsp;&nbsp;
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[[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;
[[Haynes:TechResources | <font face="trebuchet ms" style="color:#000000"> '''Lab Resources''' </font>]] &nbsp;&nbsp;&nbsp;
[[Haynes:TechResources | <font face="trebuchet ms" style="color:#000000"> '''Lab Resources''' </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:Internal | <font face="trebuchet ms" style="color:#000000"> '''Personnel''' </font>]] &nbsp;&nbsp;&nbsp;
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</div>
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[[Haynes:Calendar | <font face="trebuchet ms" style="color:#000000"> '''Calendar''' </font>]]
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</div><br>
<div style="width: 1000px">
<div style="width: 1000px">
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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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<br>
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==Bioinformatics==
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* [[Haynes:GalaxyChiP | 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.
 +
* [[Haynes:ChIPDataMining1 | 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
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<br>
<br>
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* [[Haynes:Glycerol Stocks | Glycerol Stocks]] - for long term -80°C storage
* [[Haynes:Glycerol Stocks | Glycerol Stocks]] - for long term -80°C storage
* [[Haynes:TransformationPlasmids | Transformation of E. coli with plasmid DNA]]
* [[Haynes:TransformationPlasmids | Transformation of E. coli with plasmid DNA]]
-
 
==Cell Culture: Mammalian==
==Cell Culture: Mammalian==
* [[Haynes:MamCultureMedia | Media formulas]] - cell line-specific formulas
* [[Haynes:MamCultureMedia | Media formulas]] - cell line-specific formulas
-
* [[Haynes:TransfectionPlasmid | Transfection]] - Transfection of plasmid DNA into cells
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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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==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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<br><br>
-
==Protein==
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==Protein & Enzyme Assays==
* [[Haynes:Bradford | Bradford assay]] - measure protein concentration
* [[Haynes:Bradford | Bradford assay]] - measure protein concentration
 +
* [[Haynes:ELISA | ELISA assay]] - specific antibody-based quantification of protein; simpler than Western, but no protein size data
 +
* [[Haynes:Luciferase | Luciferase assay]] - Measuring firefly luciferase reporter activity with D-luciferin substrate
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<br><br>
==Real Time Quantitative PCR==
==Real Time Quantitative PCR==
-
* [[Haynes:UPLassay | Universal Probe Library (UPL) assay]]: for the Roche Light Cycler 480
+
* [[Haynes:UPLassay | Roche Universal Probe Library (UPL) assay]] - hydrolysis probe PCR for the Roche Light Cycler 480
 +
* [[Haynes:Roche480_Analysis | Roche Data Analysis]] - Tips for doing data analysis (from a user's perspective)
 +
* [[Haynes:SYBRGreen | SYBR Green assay]] - dsDNA-binding fluorescent dye-based detection
-
==Resources at OpenWetWare==
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<br><br>
 +
 
 +
==RNA & cDNA protocols==
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* [[Haynes:TRIzol_RNeasy | RNA mini prep - TRIzol/ RNeasy column]]
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* [[Haynes:cDNA_Synthesis | cDNA Synthesis]] - procedures and reaction table templates
 +
 
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<br><br>
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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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'''DNA Ladder mix, Gene Ruler 1 kb Plus''' {{hide|  
'''DNA Ladder mix, Gene Ruler 1 kb Plus''' {{hide|  
-
Formula: [0.5 ng/ 10 μL Fermentas Gene Ruler 1 kb Plus]<br>
+
Formula: [0.5 ng/ 10 μL Fermentas Gene Ruler 1 kb Plus; 1x loading dye]<br>
Volume: 1000 μL
Volume: 1000 μL
* Gene Ruler plus (0.5 ng/μL), 100 μL
* Gene Ruler plus (0.5 ng/μL), 100 μL
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}}
}}
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'''LB Broth (Lennox)''' {{hide|  
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'''[http://openwetware.org/wiki/Haynes:LBagarAmp 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
 +
* Yeast extract, 5 g
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* NaCl, 5 g
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* 1 N NaOH, 1 mL
 +
 
 +
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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}}
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'''[http://openwetware.org/wiki/Haynes:LB_liquid_media#LB_Liquid_Media LB Broth (Lennox)]''' {{hide|  
Volume: 1 L
Volume: 1 L
* Caesin tryptone, 10 g
* Caesin tryptone, 10 g
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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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'''Tris-acetate-EDTA (TAE) Buffer, 50x''' {{hide|
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Formula: [2 M Tris, 1 M acetate, 50 mM EDTA]<br>
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Volume: 500 mL
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* Tris base (FW 121.14), 121 g
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* Glacial acetic acid, 28.55 mL
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* 500 mM EDTA, 50 mL
 +
 +
Dissolve Tris base in 200 mL dH<sub>2</sub>O (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 dH<sub>2</sub>O. 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.
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}}
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</div>
</div>

Revision as of 15:12, 4 June 2014

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

   Home     Publications     People     Contact     Protocols     Lab Resources     Personnel     Calendar


Contents

Protocols

DNA Assembly


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


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.

Tris-acetate-EDTA (TAE) Buffer, 50x

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.



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