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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[http://soslab.ee.washington.edu/igem/2011/index.php/Sequencing Sequencing Instructions]
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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/KunkelMutagensis Kunkel Mutagensis]
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[https://2011.igem.org/Team:Washington/Protocols/Ligation  General Ligation 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/Transformation  General Transformation 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/Colony Colony PCR 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/Competent  Competent Cell Prep 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/Kunkel Kunkel Mutagenesis]
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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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[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of the Kunkel Mutagenesis process]
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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/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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[https://2011.igem.org/Team:Washington/Protocols/sequencing Sequencing]
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=Protocol Page=
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[https://2011.igem.org/Team:Washington/Protocols/CompDesign  Computational Protein Design]
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'''restriction digest'''
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[https://2011.igem.org/Team:Washington/Protocols/Glycerol_Stocks  Glycerol Stocks]
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10 uL DNA
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5uL buffer ( 2 for most, check NEB)
 
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.5 uL BSA
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=Make It:  Diesel Production Protocols=
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1uL enzyme 1
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[https://2011.igem.org/Team:Washington/alkanebiosynthesis Alkane Biosynthesis media and extraction]
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1uL enzyme 2
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[https://2011.igem.org/Team:Washington/alkanebiosynthesis_cloning Alkane Biosynthesis cloning]
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water to 50 uL(32.5 uL, add first)
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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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'''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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=Break It:  Gluten Destruction Protocols=
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make oligo mix with 5uL of each primer
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[https://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_Assay Whole Cell Lysate Assay]
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PCR reaction:
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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 phusion
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.5uL oligo mix
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[https://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay Enzyme Assay]
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1uL first oligo
 
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1uL last oligo
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=Make It: iGEM Toolkits=
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5uL buffer
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[https://2011.igem.org/Team:Washington/Protocols/Cyto. Cytometry Protocol]
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1uL dnTP
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[https://2011.igem.org/Team:Washington/Protocols/Elect. Electroporation (Transformation)]
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dH20 to 50uL
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]
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'''Ligation'''
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Purif. Gibson Purification]
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7uL insert
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[https://2011.igem.org/Team:Washington/Protocols/High_PCR High-Yield PCR (Full-Gene Assembly)]
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1uL vector
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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 buffer
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[https://2011.igem.org/Team:Washington/Protocols/Induc_studies. Induction Studies of Proteins Fusions (mam-sfGFP)]
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1uL T4 ligase
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[https://2011.igem.org/Team:Washington/Protocols/pGA. pGA Vector Assay]
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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/PBS. PBS Stock Protocol]
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[https://2011.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]
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'''Colony PCR with Green tag'''
 
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Master mix(7ul):
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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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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

[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.)