Team:Washington/alkanebiosynthesis

From 2011.igem.org

(Difference between revisions)
(Long Term Stocks to Prepare)
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*1M  MgSO4
*1M  MgSO4
*0.1M FeCl3-6H2O
*0.1M FeCl3-6H2O
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==='''100mL M9 minGlucose Media Prep'''===
==='''100mL M9 minGlucose Media Prep'''===
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*100uL of FeCl3-6H2O
*100uL of FeCl3-6H2O
*100uL of MgSO4
*100uL of MgSO4
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*5uL of Each Trace Metal (do not mix together before, they crash out)
 
-
 
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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Aliquot 0.7mL into culture tubes and add appropriate antibiotic (1microL of Kan or Chlor)
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==='''General Production Protocol'''===
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Pick colonies and inoculate cultures, but DO NOT eject the tip into the tube
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-
 
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Grow cultures for 40+ hrs at 37degC (if inducible add 1mM IPTG at and OD600 of ~1.0)
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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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*Grow 5mL of cells in Terrific Broth with Antibiotics overnight to saturation in 14mL culture tube
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*Measure OD (should be roughly 1.5)
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*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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*Spin down cells at 4000rpm for 10 minutes
 +
*Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL eppindorf tube
 +
*Spin down cells at 4000rpm for 10 minutes
 +
*Resuspend in 0.7mL of Production Media with Antibiotic
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*Transfer to a 24mL 13x250
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*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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*Remove 200uL of the top Ethyl Acetate layer into a glass vial with insert
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*Run sample on GC-MS and identify Alkanes
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Remove 200uL of the top Ethyl Acetate layer into a glass vial with insert
 
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Run sample on GC-MS and identify Alkanes
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==='''GCMS Settings and Information'''===

Revision as of 01:53, 23 September 2011

Contents

Microbial Alkane Production Protocol

Current Protocol for 100mL of Media

Adopted From Supplemental Information In 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 eppindorf tube
  • Spin down cells at 4000rpm for 10 minutes
  • Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL eppindorf 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 eppindorf 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


GCMS Settings and Information