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Team:Groningen/Notebook/10 September 2009
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Wet
GVP Cluster
O.n. precultures
- → All tubes for plasmid isolation (pLacI (pSB1A2)(no.1 and no.2), pSB2K3 (12J)(2x) and pSB2K3 (12L)(2x) showed growth. The pSB2K3 had less growth (and smaller pellet) than the other tubes.
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 was collected in a 2.0mL cup (tubes from pSB2K3 12J and 12L 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 |
| pLacI no.1 (pSB1A2) | 66.3 | 1.86 | 1.82 | x | Yes (SpeI/PstI) |
| pLacI no.2 (pSB1A2) | 47.1 | 1.82 | 1.45 | x | Yes (SpeI/PstI) |
| pSB2K3 (12J) | 142.9 | 1.89 | 2.31 | x | Yes (EcoRI/PstI) |
| pSB2K3 (12L) | 337.1 | 1.87 | 2.35 | x | Yes (EcoRI/PstI) |
Restriction for Assembly
The plasmids from the o.n. precultures were cut with PstI and EcoRI to cut out the entire part between the pre- and suffix.
| Plasmid | Amount μL | MQ μL | Fast digest buffer | EcoRI fast digest enzyme | XbaI fast digest enzyme | SpeI fast digest enzyme | PstI fast digest enzyme |
| pLacI no.1 (pSB1A2) | 16.0 | x | 3.0 | x | x | 1.0 | 1.0 |
| pLacI no.1 (pSB1A2) | 16.0 | x | 3.0 | x | x | 1.0 | 1.0 |
| GVP | 4.0 | 11.0 | 3.0 | x | 1.0 | x | 1.0 |
| pSB2K3 (12J) | 8.0 | 6.0 | 3.0 | 1.0 | x | x | 1.0 |
| pSB2K3 (12L) | 4.0 | 11.0 | 3.0 | 1.0 | x | x | 1.0 |
| pZntR-GVP | 12.0 | 3.0 | 3.0 | 1.0 | x | x | 1.0 |
- → From left to right: 1kB ladder, pZntR-GVP (2x), Empty Slot, pSB2K3 12J (2x), Empty Slot, pSB2K3 12L (2x)
- → From left to right: 1kB ladder, pLacI no.1 (pSB1A2)(2x), pLacI no.2 (pSB1A2)(2x), GVP (2x), Empty Slot,
Purification
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- → In step 7 the fragments were eluted in 12μL MQ and was stored on ice until use.
Concentration
Transporters
Metal Accumulation
Vectors
Dry
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