Team:Washington/alkanebiosynthesis

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{{Template:Team:Washington/Templates/Top}}
{{Template:Team:Washington/Templates/Top}}
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=Microbial Alkane Production Protocol=
=Microbial Alkane Production Protocol=
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__TOC__
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==Current Protocol for 100mL of Media==
==Current Protocol for 100mL of Media==
Adopted From Supplemental Information In [http://www.sciencemag.org/content/329/5991/559.full Microbial Biosynthesis of Alkanes Science Report]
Adopted From Supplemental Information In [http://www.sciencemag.org/content/329/5991/559.full Microbial Biosynthesis of Alkanes Science Report]
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==='''Long Term Stocks to Prepare'''===
 
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==='''Long Term Stocks to Prepare'''===
Store at room temperature unless otherwise noted
Store at room temperature unless otherwise noted
*1L of 1M Bis‐Tris (pH 7.25)
*1L of 1M Bis‐Tris (pH 7.25)
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*1M  MgSO4
*1M  MgSO4
*0.1M FeCl3-6H2O
*0.1M FeCl3-6H2O
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Once media is prepared sterile filter into a pre-sterilized glass bottle
Once media is prepared sterile filter into a pre-sterilized glass bottle
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==='''General Production Protocol'''===
==='''General Production Protocol'''===
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*Measure OD (should be roughly 1.5)
*Measure OD (should be roughly 1.5)
*Spin down cells at 4000rpm for 10 minutes
*Spin down cells at 4000rpm for 10 minutes
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* Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL eppindorf tube
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*Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL Eppendorf tube
*Spin down cells at 4000rpm for 10 minutes
*Spin down cells at 4000rpm for 10 minutes
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*Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL eppindorf tube
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*Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL Eppendorf tube
*Spin down cells at 4000rpm for 10 minutes
*Spin down cells at 4000rpm for 10 minutes
*Resuspend in 0.7mL of Production Media with Antibiotic
*Resuspend in 0.7mL of Production Media with Antibiotic
*Transfer to a 24mL 13x250
*Transfer to a 24mL 13x250
*Seal the top with alluminum foil and grow at 37degC for 48 hours
*Seal the top with alluminum foil and grow at 37degC for 48 hours
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*Extract by adding 0.7mL of EthylAcetate, vortex, transfer to an eppindorf tube, and spin at max speed for 1minute.
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*Extract by adding 0.7mL of EthylAcetate, vortex, transfer to an Eppendorf tube, and spin at max speed for 1minute.
*Remove 200uL of the top Ethyl Acetate layer into a glass vial with insert
*Remove 200uL of the top Ethyl Acetate layer into a glass vial with insert
*Run sample on GC-MS and identify Alkanes
*Run sample on GC-MS and identify Alkanes
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==='''GCMS Settings and Information'''===
 

Latest revision as of 00:20, 29 September 2011


Microbial Alkane Production Protocol

Contents


Current Protocol for 100mL of Media

Adopted From Supplemental Information In [http://www.sciencemag.org/content/329/5991/559.full Microbial Biosynthesis of Alkanes Science Report]


Long Term Stocks to Prepare

Store at room temperature unless otherwise noted

  • 1L of 1M Bis‐Tris (pH 7.25)
  • 10mL of 1mg/mL Thiamine (store at -20 in 1mL aliquots)
  • 10mL of 10% Triton X-100
  • 1M MgSO4
  • 0.1M FeCl3-6H2O


100mL M9 minGlucose Media Prep

ADD IN ORDER, make sure you have a sterilized Erlenmeyer flask for the initial mixing and a sterilized bottle to sterile filter into

Constantly mix using a stir bar

  • 75mL ddiH2O
  • 3g glucose (Final 3%, 100% = 1g/mL)
  • 0.6g Na2HPO4
  • 0.3g KH2PO4
  • 0.05g NaCl
  • 0.2g NH4Cl
  • 20mL of 1M Bis-Tris (pH 7.25)
  • 1mL of 10% Triton
  • 100uL of 1mg/mL thiamine
  • 100uL of FeCl3-6H2O
  • 100uL of MgSO4

Once media is prepared sterile filter into a pre-sterilized glass bottle


General Production Protocol

  • Grow 5mL of cells in Terrific Broth with Antibiotics overnight to saturation in 14mL culture tube
  • Measure OD (should be roughly 1.5)
  • Spin down cells at 4000rpm for 10 minutes
  • Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL Eppendorf tube
  • Spin down cells at 4000rpm for 10 minutes
  • Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL Eppendorf tube
  • Spin down cells at 4000rpm for 10 minutes
  • Resuspend in 0.7mL of Production Media with Antibiotic
  • Transfer to a 24mL 13x250
  • Seal the top with alluminum foil and grow at 37degC for 48 hours
  • Extract by adding 0.7mL of EthylAcetate, vortex, transfer to an Eppendorf tube, and spin at max speed for 1minute.
  • Remove 200uL of the top Ethyl Acetate layer into a glass vial with insert
  • Run sample on GC-MS and identify Alkanes