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Team:Groningen/Notebook/17 September 2009
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[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
Planning
- → TODO work out the wiki page for GVP
- → DONE make a doodle for presentation planning (1-19 oct.)
- → TODO media attention
- → DONE place an ethics survey link on twitter
- → TODO clone pArsR-GVP into pSB2K3
- → TODO clone repeat out of GVP cluster
- → TODO make glycerol stocks of constructs
- → TODO enter info on part registry
Cultures
The overnight cultures with LB-amp100 medium of colonies E.coli TOP10 with pNL29 (3x), and pArsR-GVP (1-3) all showed expected growth of bacteria.
Plasmid isolation
Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids 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 (8mL for pNL29) was collected in a 2.0mL cup (tubes from pArsR-GVP, GVP and pNL29 were combined), 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
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pArsR-GVP (pSB2K3, no.1) | 40.6 | 1.89 | 1.77 | x | Yes (EcoRI/PstI) |
| pArsR-GVP (pSB2K3, no.2) | 47.3 | 1.87 | 2.01 | x | Yes (EcoRI/PstI) |
| pArsR-GVP (pSB2K3, no.3) | 40.4 | 1.89 | 1.64 | x | Yes (EcoRI/PstI) |
| pNL29 | 26.4 | 2.10 | 1.64 | x | Yes (MvaI/XhoI) |
Restriction for Control and Assembly
The plasmids from the o.n. precultures of pArsR-GVP and pNL29 were cut with PstI/EcoRI and MvaI/XhoI to cut out the entire part between the pre- and suffix and wanted fragment.
| Plasmid | Amount μL | MQ μL | Fast digest buffer | EcoRI fast digest enzyme | PstI fast digest enzyme |
| pArsR-GVP no.1 | 16.0 | x | 3.0 | 1.0 | 1.0 |
| pArsR-GVP no.2 | 16.0 | x | 3.0 | 1.0 | 1.0 |
| pArsR-GVP no.2 | 16.0 | x | 3.0 | 1.0 | 1.0 |
| Plasmid | Amount μL | MQ μL | R-digest buffer | MvaI digest enzyme | XhoI digest enzyme |
| pNL29 | 16.0 | x | 3.0 | 1.0 | 1.0 |
- → From left to right: 1kB ladder, pNL29 (orange dye), pNL29 (blue dye), pArsR-GVP no.1, pArsR-GVP no.2, pArsR-GVP no.3
Transporters
Metal Accumulation
Vectors
Dry
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