Team:Washington/Protocols

From 2011.igem.org

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<center><big><big><big><big>'''Protocols'''</big></big></big></big></center><br><br>
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=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]
[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]
[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/ Gibson Reaction]
 
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[https://2011.igem.org/Team:Washington/Protocols/ Gibson Purification]
 
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=Gibson Vectors (pGB) protocols=
 
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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]
 
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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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=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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