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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[https://2011.igem.org/Team:Washington/Protocols/PCR  General PCR Protocol]
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[https://2011.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagensis]
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[https://2011.igem.org/Team:Washington/Protocols/Digestion  General Digestion 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/Ligation  General Ligation Protocol]
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[https://2011.igem.org/Team:Washington/Protocols/plate_expression 96 Well Plate Protein Expression]
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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/DoubleDigest Double Digest]
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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/Projects:Biofuels/AlkaneBiosynthesis Alkane Biosynthesis Protocol]
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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/1L_NiNTA_Expression_Purification Standard 1L Expression Purification]
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[https://2011.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagenesis]
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[http://soslab.ee.washington.edu/igem/2011/index.php/Protocols/GeneAssembly Gene Assembly With Oligos]
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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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[https://2011.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]
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=Protocol Page=
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[https://2011.igem.org/Team:Washington/Protocols/sequencing Sequencing]
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'''restriction digest'''
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[https://2011.igem.org/Team:Washington/Protocols/CompDesign  Computational Protein Design]
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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/Glycerol_Stocks  Glycerol Stocks]
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.5 uL BSA
 
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1uL enzyme 1
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=Make It:  Diesel Production Protocols=
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1uL enzyme 2
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[https://2011.igem.org/Team:Washington/alkanebiosynthesis Alkane Biosynthesis media and extraction]
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water to 50 uL(32.5 uL, add first)
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[https://2011.igem.org/Team:Washington/alkanebiosynthesis_cloning Alkane Biosynthesis cloning]
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'''oligo assembly by PCR'''
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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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resuspend oligos with water, amount of water= concentration(in nm)*10 in uL
 
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make oligo mix with 5uL of each primer
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=Break It:  Gluten Destruction Protocols=
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PCR reaction:
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[https://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_Assay Whole Cell Lysate Assay]
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1uL phusion
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.5uL oligo mix
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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 first oligo
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[https://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay Enzyme Assay]
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1uL last oligo
 
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5uL buffer
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=Make It: iGEM Toolkits=
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1uL dnTP
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[https://2011.igem.org/Team:Washington/Protocols/Cyto. Cytometry Protocol]
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dH20 to 50uL
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[https://2011.igem.org/Team:Washington/Protocols/Elect. Electroporation (Transformation)]
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'''Ligation'''
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]
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7uL insert
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Purif. Gibson Purification]
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1uL vector
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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 buffer
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[https://2011.igem.org/Team:Washington/Protocols/Plas_DNA. Isolation of Plasmid DNA (miniprep)]
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1uL T4 ligase
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[https://2011.igem.org/Team:Washington/Protocols/Induc_studies. Induction Studies of Proteins Fusions (mam-sfGFP)]
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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/pGA. pGA Vector Assay]
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'''Colony PCR with Green tag'''
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[https://2011.igem.org/Team:Washington/Protocols/PBS. PBS Stock Protocol]
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Master mix(7ul):
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[https://2011.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]
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1ul 10uM forword primer
 
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1ul 10uM reverse Primer
 
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5ul 2x Green tag
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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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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.)