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Team:Groningen/Notebook/14 September 2009
From 2009.igem.org
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===Metal Accumulation=== | ===Metal Accumulation=== | ||
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===Vectors=== | ===Vectors=== | ||
Revision as of 12:34, 14 September 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
Observations
- Plasmids with a inducible copy number are best grown on agar plates without IPTG (probably the high amount of plasmid to be produced is to much), and growth in cultures is best done with induction by IPTG (better growth, higher density was observed).
- The test of both the new competent cells and ampicillin batch seem to be positive. On the negative plates no/small growth was observed, and on the positive plates large number of expected red colonies.
Plasmid isolation
Plasmid isolation was performed on the pellets of E.coli TOP10 plasmids (made on saterday and stored in -20box) 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 5mL 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
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pSB2K3-pZntR-GVP (12L, no.1) | 46.3 | 1.96 | 2.67 | fridge | Yes (EcoRI/PstI) |
| pSB2K3-pZntR-GVP (12L, no.2) | 40.9 | 1.89 | 2.85 | fridge | Yes (EcoRI/PstI) |
| pSB2K3-pCueO-GVP (12L, no.1) | 50.9 | 1.90 | 2.43 | fridge | Yes (EcoRI/PstI) |
| pSB2K3-pCueO-GVP (12L, no.2) | 45.2 | 1.93 | 2.80 | fridge | Yes (EcoRI/PstI) |
Restriction for Assembly
The plasmids from the o.n. precultures of pZntR-GVP and pCueO-GVP were cut with EcoRI/PstI to cut out the entire part between the pre- and suffix, which should give bands of ~6000bp and ~4450bp as a control. The GVP BioBrick plasmid was cut with XbaI/PstI to create fragment of GVP for further work in repeat removal.
| Plasmid | Amount μL | MQ μL | Fast digest buffer | EcoRI fast digest enzyme | XbaI fast digest enzyme | SpeI fast digest enzyme | PstI fast digest enzyme |
| pZntR-GVP no.1 (pSB2K3) | 10.0 | 5.0 | 3.0 | 1.0 | x | x | 1.0 |
| pZntR-GVP no.2 (pSB2K3) | 10.0 | 5.0 | 3.0 | 1.0 | x | x | 1.0 |
| pCueO-GVP no.1 (pSB2K3) | 10.0 | 5.0 | 3.0 | 1.0 | x | x | 1.0 |
| pCueO-GVP no.2 (pSB2K3) | 10.0 | 5.0 | 3.0 | 1.0 | x | x | 1.0 |
| GVP (J61035) | 2.5 | 12.5 | 3.0 | x | 1.0 | x | 1.0 |
| GVP (J61035) | 2.5 | 12.5 | 3.0 | x | 1.0 | x | 1.0 |
- → From left to right: 1kB ladder, pZntR-GVP no.1, pZntR-GVP no.2, pCueO-GVP no.1, pCueO-GVP no.2, Empty Slot, GVP (4x)
Transporters
Metal Accumulation
HmtA New pcr attemt. PCR1 PCR2 F1 and 1850 and 1820
Vectors
Dry
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