Team:Washington/Protocols

From 2011.igem.org

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__NOTOC__
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INSERT INFO HERE... LOOK AT OTHER PAGES FOR EXAMPLES OF FORMATING AND INSERTING PICTURES.  CLICK THE EDIT BUTTON ON THE UPPER LEFT SIDE OF THE PAGE AFTER YOU HAVE SIGNED IN.
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<center><big><big><big><big>'''Protocols'''</big></big></big></big></center><br><br>
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[https://2011.igem.org/Team:Washington/Alkanes/Protocols/Example_1 Example 1]
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=General Protocols=
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[https://2011.igem.org/Team:Washington/Alkanes/Protocols/Example_2 Example 2]
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[https://2011.igem.org/Team:Washington/Protocols/gel_electrophoresis General Agarose Gel Electrophoresis]
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[http://soslab.ee.washington.edu/igem/2011/index.php/Sequencing Sequencing Instructions]
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[http://soslab.ee.washington.edu/igem/2011/index.php/KunkelMutagensis Kunkel Mutagensis]
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[https://2011.igem.org/Team:Washington/Protocols/PCR  General PCR Protocol]
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[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of how Kunkel Mutagensis works]
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[https://2011.igem.org/Team:Washington/Protocols/Digestion  General Digestion Protocol]
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[http://soslab.ee.washington.edu/igem/2011/index.php/PlateExpression 96 Well Plate Protein Expression]
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[https://2011.igem.org/Team:Washington/Protocols/Ligation General Ligation Protocol]
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[http://soslab.ee.washington.edu/igem/2011/index.php/DoubleDigest Double Digest]
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[https://2011.igem.org/Team:Washington/Protocols/Transformation  General Transformation Protocol]
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[http://soslab.ee.washington.edu/igem/2011/index.php/Projects:Biofuels/AlkaneBiosynthesis Alkane Biosynthesis Protocol]
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[https://2011.igem.org/Team:Washington/Protocols/Colony Colony PCR Protocol]
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[http://soslab.ee.washington.edu/igem/2011/index.php/Protocols/1L_NiNTA_Expression_Purification Standard 1L Expression Purification]
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[https://2011.igem.org/Team:Washington/Protocols/Competent  Competent Cell Prep Protocol]
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[http://soslab.ee.washington.edu/igem/2011/index.php/Protocols/GeneAssembly Gene Assembly With Oligos]
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[https://2011.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagenesis]
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[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of the Kunkel Mutagenesis process]
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[https://2011.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]
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=Protocol Page=
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[https://2011.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]
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'''restriction digest'''
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[https://2011.igem.org/Team:Washington/Protocols/sequencing Sequencing]
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10 uL DNA
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5uL buffer ( 2 for most, check NEB)
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[https://2011.igem.org/Team:Washington/Protocols/CompDesign  Computational Protein Design]
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.5 uL BSA
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[https://2011.igem.org/Team:Washington/Protocols/Glycerol_Stocks  Glycerol Stocks]
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1uL enzyme 1
 
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1uL enzyme 2
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=Make It:  Diesel Production Protocols=
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water to 50 uL(32.5 uL, add first)
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[https://2011.igem.org/Team:Washington/alkanebiosynthesis Alkane Biosynthesis media and extraction]
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'''oligo assembly by PCR'''
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[https://2011.igem.org/Team:Washington/alkanebiosynthesis_cloning Alkane Biosynthesis cloning]
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resuspend oligos with water, amount of water= concentration(in nm)*10 in uL
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[https://2011.igem.org/Team:Washington/Protocols/redesign_cell_lysate_assay Cell Lysate Assay by Decarbonylase Redesign Team]
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make oligo mix with 5uL of each primer
 
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PCR reaction:
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=Break It: Gluten Destruction Protocols=
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1uL phusion
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.5uL oligo mix
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[https://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_Assay Whole Cell Lysate Assay]
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1uL first oligo
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[https://2011.igem.org/Team:Washington/Protocols/50mL_Scale Small Scale (50mL) Protein Expression and Purification]
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1uL last oligo
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[https://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay Enzyme Assay]
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5uL buffer
 
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1uL dnTP
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=Make It: iGEM Toolkits=
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dH20 to 50uL
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[https://2011.igem.org/Team:Washington/Protocols/Cyto. Cytometry Protocol]
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'''Ligation'''
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[https://2011.igem.org/Team:Washington/Protocols/Elect. Electroporation (Transformation)]
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7uL insert
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]
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1uL vector
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Purif. Gibson Purification]
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1uL T4 ligase buffer
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[https://2011.igem.org/Team:Washington/Protocols/High_PCR High-Yield PCR (Full-Gene Assembly)]
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1uL T4 ligase
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[https://2011.igem.org/Team:Washington/Protocols/Plas_DNA. Isolation of Plasmid DNA (miniprep)]
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incubate at <s>37C</s> no. **Note that NEB website recommends 16C - room temp for ligations, we do ours at RT and it works well. 1 hour.
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[https://2011.igem.org/Team:Washington/Protocols/Induc_studies. Induction Studies of Proteins Fusions (mam-sfGFP)]
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[https://2011.igem.org/Team:Washington/Protocols/pGA. pGA Vector Assay]
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[https://2011.igem.org/Team:Washington/Protocols/PBS. PBS Stock Protocol]
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'''Colony PCR with Green tag'''
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[https://2011.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]
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Master mix(7ul):
 
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1ul 10uM forword primer
 
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1ul 10uM reverse Primer
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=Wiki Design=
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[https://2011.igem.org/Team:Washington/Protocols/Wiki_Design Wiki Design Tools (Wiki Markup, WikiDust, etc.)]
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5ul 2x Green tag
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Cell water(3ul): Pick one colony from the plate and mix with 10ul of ddH2O to make 10ul of cell water
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Reaction = Master mix(7ul) + Cell water(3ul) = 10ul per tube
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Use program "Colony" & change the extention time (1min per kb)
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'''Heat Shock Transformation'''
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2 ul ligation
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20 ul cells
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Ice 20 minutes
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Heat shock at 42C for 1 minute
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Ice 2 minutes
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Prepare 200 ul of TB (no anti) and transformed cells in culture tube
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Incubate at 37C for 1 hour
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Plate cells
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Latest revision as of 02:10, 29 September 2011


Protocols


General Protocols

General Agarose Gel Electrophoresis

General PCR Protocol

General Digestion Protocol

General Ligation Protocol

General Transformation Protocol

Colony PCR Protocol

Competent Cell Prep Protocol

Kunkel Mutagenesis

[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of the Kunkel Mutagenesis process]

Standard 1L Expression Purification

Gene Assembly With Oligos

Sequencing

Computational Protein Design

Glycerol Stocks


Make It: Diesel Production Protocols

Alkane Biosynthesis media and extraction

Alkane Biosynthesis cloning

Cell Lysate Assay by Decarbonylase Redesign Team


Break It: Gluten Destruction Protocols

Whole Cell Lysate Assay

Small Scale (50mL) Protein Expression and Purification

Enzyme Assay


Make It: iGEM Toolkits

Cytometry Protocol

Electroporation (Transformation)

Gibson Cloning/Assembly

Gibson Purification

High-Yield PCR (Full-Gene Assembly)

Isolation of Plasmid DNA (miniprep)

Induction Studies of Proteins Fusions (mam-sfGFP)

pGA Vector Assay

PBS Stock Protocol

Preparation of Overnight Cultures


Wiki Design

Wiki Design Tools (Wiki Markup, WikiDust, etc.)