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]]
 +
* [[Haynes:PCR_clip_clone | PCR Clip and Clone]] - robust method to clone a PCR-amplified insert, minimizing loss of insert yeild
 +
* [[Haynes:TypeIIS_Assembly | Type IIS Assembly]] - similar to Golden Gate, Golden Braid, and Moclo
 +
* [[Haynes:LCR_Assembly | Ligase Cycling Reaction]] - ligation of blunt-ended fragments guided by overlapping oligo "bridges"
 +
 +
<br>
 +
 +
==Bioinformatics==
 +
* [[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
 +
* [[Haynes:GOEnrichment | Gene Ontology Term Enrichment]] - How to compute which standardized terms (GO terms) for cell behavior/ biology are significantly over-represented in a user-defined list of genes
 +
<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:ThawingCells | Thawing Cells]] - starting a new culture from a -150°C frozen stock vial
 +
* [[Haynes:AdvancingCells | Advancing Cells]] - transferring a new thawed culture into a larger growth vessel
 +
* [[Haynes:SplittingCells | Splitting Cells]] - splitting or passaging cells
* [[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
 +
* [[Haynes:CryopreservationMamCells | Cryopreserving Cells]] - Preparing frozen cell stocks
 +
==Imaging: Mammalian==
-
==Protein==
+
'''Flow Cytometry'''
-
* [[Haynes:Bradford | Bradford assay]] - measure protein concentration
+
* [[Haynes:FCMamalian_FluorGene | Detecting Fluorescent Protein Expression]]
 +
* [[Haynes:ICCSaponin | Intracellular Protein Staining with Saponin Permeabilization]] - how to detect cytoplasmic proteins using antibodies
 +
'''Microscopy'''
 +
* [[Haynes:Mamalian_IFC | Immunocytology]] - staining fixed cells  with antibodies
 +
 +
<br><br>
 +
 +
==Protein & Enzyme Assays==
 +
* [[Haynes:Bradford | Bradford assay]] - measure extracted protein concentration
 +
* [[Haynes:ELISA | ELISA assay]] - specific antibody-based quantification of protein; simpler than Western, but no protein size data
 +
* [[Haynes:ExtFluorProtein | Fluorescent Protein]] - measure extracted fluorescent protein levels
 +
* [[Haynes:Luciferase | Luciferase assay]] - measuring firefly luciferase reporter activity with D-luciferin substrate
 +
* [[Haynes:ProteinExtraction | Protein Extraction]] - extracting proteins from mammalian cells
 +
* [[Haynes:PAGE | PAGE]] - polyacrylamide gel electrophoresis (PAGE)
 +
* [[Haynes:WestBlot | Western Blot]] - gel-to-membrane protein transfer; detection with antibodies
 +
 +
<br><br>
==Real Time Quantitative PCR==
==Real Time Quantitative PCR==
-
* [[Haynes:UPLassay | Universal Probe Library (UPL) assay]] - for the Roche Light Cycler 480
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* [[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
 +
 
 +
<br><br>
==RNA & cDNA protocols==
==RNA & cDNA protocols==
* [[Haynes:TRIzol_RNeasy | RNA mini prep - TRIzol/ RNeasy column]]
* [[Haynes:TRIzol_RNeasy | RNA mini prep - TRIzol/ RNeasy column]]
 +
* [[Haynes:cDNA_Synthesis | cDNA Synthesis]] - procedures and reaction table templates
 +
<br><br>
==Other Resources - OpenWetWare==
==Other Resources - OpenWetWare==
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==Software Guides==
==Software Guides==
* [[Haynes:Jmol_Guide | Jmol commands: controlling PDB structure displays]]
* [[Haynes:Jmol_Guide | Jmol commands: controlling PDB structure displays]]
 +
* [http://openwetware.org/wiki/BME100_s2015:WikiHelp Wiki Editing Guide 1] - from Dr. Haynes' BME100 course
 +
* [http://openwetware.org/wiki/Simple_wiki_editing_examples Wiki Editing Guide 2] - simple examples from OWW
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}}
}}
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'''LB Broth (Lennox)''' {{hide|  
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'''LB (Lennox) Agar plates'''
 +
:[http://openwetware.org/wiki/Haynes:LBagarAmp Detailed protocol]
 +
:{{hide|
 +
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 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.
 +
}}
 +
 
 +
'''LB Broth (Lennox)'''  
 +
:[http://openwetware.org/wiki/Haynes:LB_liquid_media#LB_Liquid_Media Detailed protocol]
 +
:{{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.
}}
}}
 +
 +
'''Oligo Annealing Buffer, 10x''' {{hide|
 +
Formula: [1M NaCl; 100 mM Tris-HCl pH 7.4]<br>
 +
Volume: 1000 μL
 +
* 5M NaCl, 200 μL
 +
* 1M Tris-HCl, 100 μL
 +
* dH<sub>2</sub>O, 700 μL
 +
 +
Keep frozen at -20°C.
 +
}}
 +
 +
'''Resuspension Buffer - for plasmid minipreps''' {{hide|
 +
Formula: [25 mM Tris-HCl, 10 mM EDTA, 100 μg/mL RNaseA, pH 8.0] (based on the Sigma formulation)<br>
 +
Volume: 50 mL
 +
* 1M Tris-HCl pH ~8, 2.0 mL
 +
* 500 mM EDTA, 1.0 mL
 +
* Molecular biology grade dH<sub>2</sub>O, up to 45 mL
 +
** Adjust pH to 8.0
 +
* Molecular biology grade dH<sub>2</sub>O, up to 50 mL
 +
* 20 mg/mL RNaseA, 250 μL
 +
 +
In a 50 mL conical, add Tris-HCl and EDTA. Fill up to ~45 mL with molecular biology grade dH<sub>2</sub>O. Adjust pH to 8.0: add 10 μL acid or base, cap tightly, invert to mix, then measure pH. Repeat as needed. Fill up to 50 mL with molecular biology grade dH<sub>2</sub>O. Cap tightly and invert to mix. Keep refrigerated at 4°C.
 +
}}
 +
 +
'''Tris-acetate-EDTA (TAE) Buffer, 50x''' {{hide|
 +
Formula: [2 M Tris, 1 M acetate, 50 mM EDTA]<br>
 +
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 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.
 +
}}
 +
 +
'''Tris-glycine concentrate''' {{hide|
 +
Formula: [250 mM Tris; 1.92 M glycine; pH approx. 8.3]<br>
 +
Volume: 500 mL
 +
* Tris base (FW 121.14), 15.15 g
 +
* Glycine, 72.0 g
 +
 +
Dissolve Tris base and glycine in 200 mL dH<sub>2</sub>O (in a beaker with stirring). Transfer solution to a graduated cylinder and fill up to 500 mL with dH<sub>2</sub>O. Do not autoclave.
 +
}}
 +
 +
</div>
</div>

Current revision

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

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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
  • Gene Ontology Term Enrichment - How to compute which standardized terms (GO terms) for cell behavior/ biology are significantly over-represented in a user-defined list of genes



Cell Culture: Bacteria


Cell Culture: Mammalian

Imaging: Mammalian

Flow Cytometry

Microscopy



Protein & Enzyme Assays

  • Bradford assay - measure extracted protein concentration
  • ELISA assay - specific antibody-based quantification of protein; simpler than Western, but no protein size data
  • Fluorescent Protein - measure extracted fluorescent protein levels
  • Luciferase assay - measuring firefly luciferase reporter activity with D-luciferin substrate
  • Protein Extraction - extracting proteins from mammalian cells
  • PAGE - polyacrylamide gel electrophoresis (PAGE)
  • Western Blot - gel-to-membrane protein transfer; detection with antibodies



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

Detailed protocol

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)

Detailed protocol

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.

Resuspension Buffer - for plasmid minipreps

Formula: [25 mM Tris-HCl, 10 mM EDTA, 100 μg/mL RNaseA, pH 8.0] (based on the Sigma formulation)
Volume: 50 mL

  • 1M Tris-HCl pH ~8, 2.0 mL
  • 500 mM EDTA, 1.0 mL
  • Molecular biology grade dH2O, up to 45 mL
    • Adjust pH to 8.0
  • Molecular biology grade dH2O, up to 50 mL
  • 20 mg/mL RNaseA, 250 μL

In a 50 mL conical, add Tris-HCl and EDTA. Fill up to ~45 mL with molecular biology grade dH2O. Adjust pH to 8.0: add 10 μL acid or base, cap tightly, invert to mix, then measure pH. Repeat as needed. Fill up to 50 mL with molecular biology grade dH2O. Cap tightly and invert to mix. Keep refrigerated at 4°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.

Tris-glycine concentrate

Formula: [250 mM Tris; 1.92 M glycine; pH approx. 8.3]
Volume: 500 mL

  • Tris base (FW 121.14), 15.15 g
  • Glycine, 72.0 g

Dissolve Tris base and glycine in 200 mL dH2O (in a beaker with stirring). Transfer solution to a graduated cylinder and fill up to 500 mL with dH2O. Do not autoclave.



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