Difference between revisions of "IGEM:Harvard/2010/General Protocols"

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(Growing a Bacterial E. coli Culture)
(Microbiology)
 
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=Growing a Bacterial E. coli Culture=
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=Synthetic Biology=
E. coli is a typical bacteria used for molecular cloning. Long-term storage of BioBrick plasmids is in E. coli suspended in glycerol (500ul cells:500ul glycerol) and kept at -80˚C. Growing E. coli is performed at 37˚C, at which temperature they divide every 20 minutes.
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*[[IGEM:Harvard/2010/General Protocols/BioBricks | How Do BioBricks Work?]]
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*[[Silver: BB Strategy | Strategy for Making Parts]]
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*[[Silver: PCR | PCR]]
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*[[Silver: Oligonucleotide Inserts | Oligonucleotide Inserts]]
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*[[Silver: Site-Directed Mutagenesis | Site-Directed Mutagenesis]]
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*[[Silver: Restriction Digest | Restriction Digest]]
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*[[IGEM:Harvard/2010/General Protocols/DNApurification | DNA Gel Electrophoresis and Fragment Purification]]
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*[[IGEM:Harvard/2010/General Protocols/Nanodrop | Measuring DNA Concentration Using a Nanodrop Spectrophotometer]]
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*[[Silver: Ligation | Ligation]]
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*[[Silver: Quick & Dirty Digest & Ligation | Quick & Dirty Digest & Ligation]]
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*[[Silver: Bacterial Transformation | Bacterial Transformation]]
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*[[Silver: Plasmid Verification | Plasmid Verification]]
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*[http://labs.fhcrc.org/fero/Protocols/RNeasy_Mini_Handbook.pdf Qiagen RNeasy Plant Mini Kit]
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*[http://cshprotocols.cshlp.org/cgi/reprint/2007/4/pdb.kit23.pdf iScript cDNA Synthesis]
  
*Day1: Making streaks from glycerol stocks
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=Microbiology=
**Warm agar plate at 37˚C for 10 minutes
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*[[IGEM:Harvard/2010/General Protocols/EColi | Growing E. coli]]
**Label lid of plate with what you're streaking on it, and your name, and the date
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*[[IGEM:Harvard/2010/General Protocols/Agrobacterium | Growing Agrobacterium]]
**Locate desired glycerol stock in -80˚C freezer and, using a sterile toothpick, scrape out a tiny bit and streak on plate
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*[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium | Making Electro-Competent Agrobacterium]]
**Incubate plate overnite at 37˚C
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*[[IGEM:Harvard/2010/General Protocols/Agrobacterium Electroporation | Electroporating Agrobacterium]]
  
*Day2: Growing liquid cultures
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=Plants=
 
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*[[IGEM:Harvard/2010/General Protocols/Arabidopsis | Growing Arabidopsis]]
=Plasmid Minipreps: Qiagen Miniprep Kit=
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*[[IGEM:Harvard/2010/General Protocols/FlowerDipping | Arabidopsis transformation via Flower Dipping]]
To extract plasmid DNA from bacteria, perform a miniprep using the Qiagen miniprep protocol.
 
 
 
=DNA Digests=
 
=DNA Gel Electrophoresis and Fragment Purification=
 
=Measuring DNA Concentration using a Nanodrop Spectrophotometer=
 
=Ligation Reaction=
 
=Transformation of E. coli=
 
=Confirming the Assembly=
 
=Growing Arabidopsis=
 
=Growing an Agrobacterium Culture=
 
=Arabidopsis transformation via Flower Dipping=
 

Latest revision as of 10:54, 1 July 2010

Synthetic Biology

Microbiology

Plants