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Team:Groningen/Notebook/22 August 2009
From 2009.igem.org
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:→ {{todo}} Look at plates stored at 37C with BBa_J61002-pArsR<sup>+</sup>/pZntR<sup>+</sup>/pCueO<sup>+</sup> and use colonies to grow o.n. cultures for plasmid isolation | :→ {{todo}} Look at plates stored at 37C with BBa_J61002-pArsR<sup>+</sup>/pZntR<sup>+</sup>/pCueO<sup>+</sup> and use colonies to grow o.n. cultures for plasmid isolation | ||
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| + | '''Plates''' | ||
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| + | Showed single colony growth on plates with J61002-pME<sup>tal</sup>+RBS-RFP plasmids, and were stored in the fridge for future preculture growth. | ||
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| + | :→ The plates with high and low concentration of transformed cells showed colonies in the expected ratio. | ||
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| + | :→ On the plates for pZntR<sup>+</sup> and pCueO<sup>+</sup> no colonies were dark red, indication of ligation of the original high constitutive promoter back into the J61002 vector, or uncut plasmid which was transformed. | ||
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| + | :→ The plates with pArsR<sup>+</sup> plasmid showed only a few non-coloured colonies, mostly medium red colour, andn a few dark red colonies. It was already thought that the pArsR is a bit leaky and causes expression of RFP. | ||
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| + | '''Over Night Cultures''' | ||
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| + | :→ Both cultures of BBa_J61002-J23101 showed growth, and can be used for plasmid isolation. | ||
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| + | [[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]] | ||
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| + | '''Plasmid Purification''' | ||
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| + | Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:pSB3K3 pSB3K3] 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 and GVP with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit". | ||
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| + | * From each tube 4mL 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 30μL MQ and stored in the fridge | ||
===Transporters=== | ===Transporters=== | ||
Revision as of 09:11, 22 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 Isolate plasmids from E.coli Top10 BBa_J23101 cells
- → DONE Plate E.coli Top10 BBa_J23101 cells from o.n. culture
- → TODO
Pellet remaining cells for short storage in -20 box(not needed)
- → DONE Place the four test cultures with and without GVP on table at room temperature (make foto)
- → TODO Look at plates stored at 37C with BBa_J61002-pArsR+/pZntR+/pCueO+ and use colonies to grow o.n. cultures for plasmid isolation
Plates
Showed single colony growth on plates with J61002-pMEtal+RBS-RFP plasmids, and were stored in the fridge for future preculture growth.
- → The plates with high and low concentration of transformed cells showed colonies in the expected ratio.
- → On the plates for pZntR+ and pCueO+ no colonies were dark red, indication of ligation of the original high constitutive promoter back into the J61002 vector, or uncut plasmid which was transformed.
- → The plates with pArsR+ plasmid showed only a few non-coloured colonies, mostly medium red colour, andn a few dark red colonies. It was already thought that the pArsR is a bit leaky and causes expression of RFP.
Over Night Cultures
- → Both cultures of BBa_J61002-J23101 showed growth, and can be used for plasmid isolation.
Plasmid Purification
Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:pSB3K3 pSB3K3] 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 and GVP 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 4mL 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 30μL MQ and stored in the fridge
Transporters
Metal Accumulation
Vectors
Dry
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