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Team:Groningen/Notebook/11 August 2009
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Wet
GVP Cluster
- → DONE isolate plasmids from overnight precultures
- → DONE plate on cultures for storage in fridge (amp)
- → TODO cut plasmid and GVP containing plasmid for ligation (ligation tomorrow)
- → TODO purify wanted fragments (tomorrow)
- → DONE make o.n. precultures from plate colonies
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 J61002] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 medium] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 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 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 20μL MQ and stored in the fridge
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pJ61002-BBa_J23100 (high) | 174.6 | 1.94 | 2.40 | E-8 | No |
| pJ61002-BBa_J23106 (medium) | 149.2 | 1.95 | 2.41 | E-9 | No |
| pJ61002-BBa_J23109 (low) | 152.8 | 1.94 | 2.38 | E-10 | No |
Plates
LB-agar plates with 50 μg/mL ampicillin were freshly prepared, and one drop of each o.n. preculture was spotted and subsequently swiped three times with sterile needle. The plates were stored at 37°C overnight.
Precultures
Over night cultures in 5mL LB-amp50 medium were prepared from the following colonies:
→ E.coli TOP10 pSB1AC3-med. const. promoter - GVP (amp.) (4x) (1 to 4) → E.coli TOP10 pSB1AC3-low const. promoter - GVP (amp.) (4x) (5 to 8)
and put in the 37°C waterbath at 200 rpm.
Transporters
Metal Accumulation
Vectors
Dry
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