From 2011.igem.org

Microbial Alkane Production Protocol

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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