Team:Washington/Protocols

From 2011.igem.org

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<center><big><big><big><big>'''Protocols'''</big></big></big></big></center><br><br>
=General Protocols=
=General Protocols=
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[https://2011.igem.org/Team:Washington/Protocols/gel_electrophoresis  General Agarose Gel Electrophoresis]
[https://2011.igem.org/Team:Washington/Protocols/PCR  General PCR Protocol]
[https://2011.igem.org/Team:Washington/Protocols/PCR  General PCR Protocol]
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[https://2011.igem.org/Team:Washington/Protocols/Competent  Competent Cell Prep Protocol]
[https://2011.igem.org/Team:Washington/Protocols/Competent  Competent Cell Prep 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/Kunkel Kunkel Mutagenesis]
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[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of how Kunkel Mutagensis works]
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[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of the Kunkel Mutagenesis process]
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<!-- link is broken [https://2011.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification] >
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[https://2011.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]
[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]
[https://2011.igem.org/Team:Washington/Protocols/CompDesign  Computational Protein Design]
[https://2011.igem.org/Team:Washington/Protocols/CompDesign  Computational Protein Design]
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[https://2011.igem.org/Team:Washington/Protocols/sequencing Sequencing]
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[https://2011.igem.org/Team:Washington/Protocols/Glycerol_Stocks  Glycerol Stocks]
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=Make It:  Diesel Production Protocols=
=Make It:  Diesel Production Protocols=
[https://2011.igem.org/Team:Washington/alkanebiosynthesis Alkane Biosynthesis media and extraction]
[https://2011.igem.org/Team:Washington/alkanebiosynthesis Alkane Biosynthesis media and extraction]
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[https://2011.igem.org/Team:Washington/alkanebiosynthesis_cloning Alkane Biosynthesis cloning]
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[https://2011.igem.org/Team:Washington/Protocols/redesign_cell_lysate_assay Cell Lysate Assay by Decarbonylase Redesign Team]
=Break It:  Gluten Destruction Protocols=
=Break It:  Gluten Destruction Protocols=
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[https://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_Assay Cell Lysate Assay]
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[https://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_Assay Whole Cell Lysate Assay]
[https://2011.igem.org/Team:Washington/Protocols/50mL_Scale Small Scale (50mL) Protein Expression and Purification]
[https://2011.igem.org/Team:Washington/Protocols/50mL_Scale Small Scale (50mL) Protein Expression and Purification]
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[https://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay Enzyme Assay]
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=Make It: Magnetosome Protocols=
 
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Reaction]
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=Make It: iGEM Toolkits=
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Purif. Gibson Purification]
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[https://2011.igem.org/Team:Washington/Protocols/Cyto. Cytometry Protocol]
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=Gibson Vectors (pGB) protocols=
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[https://2011.igem.org/Team:Washington/Protocols/Elect. Electroporation (Transformation)]
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Purif. Gibson Purification]
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[https://2011.igem.org/Team:Washington/Protocols/High_PCR High-Yield PCR (Full-Gene Assembly)]
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[https://2011.igem.org/Team:Washington/Protocols/Plas_DNA. Isolation of Plasmid DNA (miniprep)]
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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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[https://2011.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]
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=Wiki Design=
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Check out or add wiki design tools here:
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[https://2011.igem.org/Team:Washington/Protocols/Wiki_Design Wiki Design Tools (Wiki Markup, WikiDust, etc.)]
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[https://2011.igem.org/Team:Washington/Protocols/Wiki_Design Wiki Design]
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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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.5 uL BSA
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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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1uL vector
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1uL T4 ligase buffer
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1uL T4 ligase
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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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'''Colony PCR with Green tag'''
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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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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

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.)