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/Digestion  General Digestion Protocol]
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[https://2011.igem.org/Team:Washington/Protocols/Wiki_Design Wiki Design]
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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/Transformation  General Transformation 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/Colony Colony 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/Competent  Competent Cell Prep 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/Kunkel Kunkel Mutagenesis]
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[https://2011.igem.org/Team:Washington/alkanebiosynthesis Alkane Biosynthesis media and extraction]
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[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of the Kunkel Mutagenesis process]
[https://2011.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]
[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]
[https://2011.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]
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[https://2011.igem.org/Team:Washington/Protocols/sequencing Sequencing]
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[https://2011.igem.org/Team:Washington/Protocols/CompDesign  Computational Protein Design]
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=Protocol Page=
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[https://2011.igem.org/Team:Washington/Protocols/Glycerol_Stocks  Glycerol Stocks]
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'''restriction digest'''
 
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10 uL DNA
 
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5uL buffer ( 2 for most, check  http://www.neb.com/nebecomm/DoubleDigestCalculator.asp)
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=Make It: Diesel Production Protocols=
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.5 uL BSA
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[https://2011.igem.org/Team:Washington/alkanebiosynthesis Alkane Biosynthesis media and extraction]
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1uL enzyme 1
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1uL enzyme 2
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water to 50 uL(32.5 uL, add first)
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'''oligo assembly by PCR'''
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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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PCR reaction:
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1uL phusion
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.5uL oligo mix
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1uL first oligo
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1uL last oligo
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5uL buffer
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1uL dnTP
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dH20 to 50uL
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'''Ligation'''
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7uL insert
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[https://2011.igem.org/Team:Washington/alkanebiosynthesis_cloning Alkane Biosynthesis cloning]
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1uL vector
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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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1uL T4 ligase buffer
 
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1uL T4 ligase
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=Break It:  Gluten Destruction Protocols=
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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/Cell_Lysate_Assay Whole Cell Lysate Assay]
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'''Colony PCR with Green tag'''
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[https://2011.igem.org/Team:Washington/Protocols/50mL_Scale Small Scale (50mL) Protein Expression and Purification]
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Master mix(7ul):
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[https://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay Enzyme Assay]
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1ul 10uM forword primer
 
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1ul 10uM reverse Primer
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=Make It: iGEM Toolkits=
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5ul 2x Green tag
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[https://2011.igem.org/Team:Washington/Protocols/Cyto. Cytometry Protocol]
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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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[https://2011.igem.org/Team:Washington/Protocols/Elect. Electroporation (Transformation)]
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Reaction = Master mix(7ul) + Cell water(3ul) = 10ul per tube
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]
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Use program "Colony" & change the extention time (1min per kb)
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Purif. Gibson Purification]
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'''Heat Shock Transformation'''
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[https://2011.igem.org/Team:Washington/Protocols/High_PCR High-Yield PCR (Full-Gene Assembly)]
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2 ul ligation
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[https://2011.igem.org/Team:Washington/Protocols/Plas_DNA. Isolation of Plasmid DNA (miniprep)]
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20 ul cells
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[https://2011.igem.org/Team:Washington/Protocols/Induc_studies. Induction Studies of Proteins Fusions (mam-sfGFP)]
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Ice 20 minutes
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[https://2011.igem.org/Team:Washington/Protocols/pGA. pGA Vector Assay]
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Heat shock at 42C for 1 minute
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[https://2011.igem.org/Team:Washington/Protocols/PBS. PBS Stock Protocol]
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Ice 2 minutes
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[https://2011.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]
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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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=Wiki Design=
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[https://2011.igem.org/Team:Washington/Protocols/Wiki_Design Wiki Design Tools (Wiki Markup, WikiDust, etc.)]

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.)

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