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Team:Groningen/Notebook/14 August 2009
From 2009.igem.org
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:→ {{done}} make glycerol stocks for pSB1AC3-BBa_J23106-BBa_I750016 and pSB1AC3-BBa_J23109-BBa_I750016 (two o.n. cultures of each are grown) | :→ {{done}} make glycerol stocks for pSB1AC3-BBa_J23106-BBa_I750016 and pSB1AC3-BBa_J23109-BBa_I750016 (two o.n. cultures of each are grown) | ||
:→ {{done}} isolate plasmid J61002-BBa_J23100, J61002-BBa_J23106, and J61002-BBa_J23109 from o.n. cultures | :→ {{done}} isolate plasmid J61002-BBa_J23100, J61002-BBa_J23106, and J61002-BBa_J23109 from o.n. cultures | ||
| - | :→ {{ | + | :→ {{done}} make a planning for next week |
'''Plates''' | '''Plates''' | ||
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Showed single colony growth on plates with pSB3K3-H/L-GVP plasmids, and were stored in the fridge for future preculture growth. | Showed single colony growth on plates with pSB3K3-H/L-GVP plasmids, and were stored in the fridge for future preculture growth. | ||
| - | :→ The plates with pSB1AC3-H-GVP plasmid showed no growth, and were stored at | + | :→ The plates with pSB1AC3-H-GVP plasmid showed no growth, and were stored at 37°C for an additional 4 hours to be sure. Still no growth after addition incubation at 37°C, can be due to high energy cost of vesicle production resulting in the death of the cells! |
'''Over Night Cultures''' | '''Over Night Cultures''' | ||
| - | :→ All cultures showed growth, and can be used for plasmid isolation and glycerol stocks. | + | :→ All cultures showed growth, and can be used for plasmid isolation and glycerol stocks. From each culture 800uL was added to 300uL 87% glycerol and frozen in liquid nitrogen. Stocks were stored at position 21 to 24 in the -80°C fridge. |
[[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]] | [[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]] | ||
Revision as of 09:49, 14 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
- → DONE make glycerol stocks for pSB1AC3-BBa_J23106-BBa_I750016 and pSB1AC3-BBa_J23109-BBa_I750016 (two o.n. cultures of each are grown)
- → DONE isolate plasmid J61002-BBa_J23100, J61002-BBa_J23106, and J61002-BBa_J23109 from o.n. cultures
- → DONE make a planning for next week
Plates
Showed single colony growth on plates with pSB3K3-H/L-GVP plasmids, and were stored in the fridge for future preculture growth.
- → The plates with pSB1AC3-H-GVP plasmid showed no growth, and were stored at 37°C for an additional 4 hours to be sure. Still no growth after addition incubation at 37°C, can be due to high energy cost of vesicle production resulting in the death of the cells!
Over Night Cultures
- → All cultures showed growth, and can be used for plasmid isolation and glycerol stocks. From each culture 800uL was added to 300uL 87% glycerol and frozen in liquid nitrogen. Stocks were stored at position 21 to 24 in the -80°C fridge.
Plasmid Purification
Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 medium] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 low] constitutive promoters with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit".
- From each tube 8mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
- Plasmids were eluted with 50μL MQ and stored in the fridge
Concentration
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| BBa_J61002-BBa_J23100 (high+RFP) | 288.1 | 1.90 | 2.33 | D-8 | No |
| BBa_J61002-BBa_J23106 (med.+RFP) | 162.2 | 1.93 | 2.31 | D-9 | No |
| BBa_J61002-BBa_J23109 (low+RFP) | 114.2 | 1.93 | 2.28 | D-10 | No |
Transporters
Metal Accumulation
Vectors
Dry
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