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Team:Washington/alkanebiosynthesis
From 2011.igem.org
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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'''=== | ||
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 | ||
| - | * Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL | + | *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 1mL of sterile ddiH2O and transfer to a 1.5mL | + | *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 | ||
| - | *Extract by adding 0.7mL of EthylAcetate, vortex, transfer to an | + | *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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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
