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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=Protocol Page=
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'''restriction digest'''
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[https://2011.igem.org/Team:Washington/Protocols/PCR  General PCR Protocol]
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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/Digestion  General Digestion Protocol]
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.5 uL BSA
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[https://2011.igem.org/Team:Washington/Protocols/Ligation  General Ligation Protocol]
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1uL enzyme 1
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[https://2011.igem.org/Team:Washington/Protocols/Transformation  General Transformation Protocol]
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1uL enzyme 2
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[https://2011.igem.org/Team:Washington/Protocols/Colony Colony PCR Protocol]
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water to 50 uL(32.5 uL, add first)
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[https://2011.igem.org/Team:Washington/Protocols/Competent  Competent Cell Prep Protocol]
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'''oligo assembly by PCR'''
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[https://2011.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagenesis]
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resuspend oligos with water, amount of water= concentration(in nm)*10 in uL
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[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of the Kunkel Mutagenesis process]
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make oligo mix with 5uL of each primer
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[https://2011.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]
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PCR reaction:
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[https://2011.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]
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1uL phusion
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.5uL oligo mix
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[https://2011.igem.org/Team:Washington/Protocols/sequencing Sequencing]
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1uL first oligo
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[https://2011.igem.org/Team:Washington/Protocols/CompDesign  Computational Protein Design]
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1uL last oligo
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[https://2011.igem.org/Team:Washington/Protocols/Glycerol_Stocks  Glycerol Stocks]
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5uL buffer
 
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1uL dnTP
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=Make It:  Diesel Production Protocols=
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dH20 to 50uL
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[https://2011.igem.org/Team:Washington/alkanebiosynthesis Alkane Biosynthesis media and extraction]
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'''Ligation'''
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[https://2011.igem.org/Team:Washington/alkanebiosynthesis_cloning Alkane Biosynthesis cloning]
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7uL insert
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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 vector
 
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1uL T4 ligase buffer
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=Break It:  Gluten Destruction Protocols=
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1uL T4 ligase
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[https://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_Assay Whole Cell Lysate 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/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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'''Colony PCR with Green tag'''
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=Make It: iGEM Toolkits=
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Master mix(7ul):
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[https://2011.igem.org/Team:Washington/Protocols/Cyto. Cytometry Protocol]
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1ul 10uM forword primer
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[https://2011.igem.org/Team:Washington/Protocols/Elect. Electroporation (Transformation)]
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1ul 10uM reverse Primer
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]
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5ul 2x Green tag
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[https://2011.igem.org/Team:Washington/Protocols/Gib_Purif. Gibson Purification]
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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/High_PCR High-Yield PCR (Full-Gene Assembly)]
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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/Plas_DNA. Isolation of Plasmid DNA (miniprep)]
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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/Induc_studies. Induction Studies of Proteins Fusions (mam-sfGFP)]
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'''Heat Shock Transformation'''
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[https://2011.igem.org/Team:Washington/Protocols/pGA. pGA Vector Assay]
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2 ul ligation
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[https://2011.igem.org/Team:Washington/Protocols/PBS. PBS Stock Protocol]
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20 ul cells
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[https://2011.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]
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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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=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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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.)