From 2009.igem.org
Wet
GVP Cluster
- → DONE isolate plasmids from o.n. cultures
- → DONE run gel to check plasmid size (should be ~9000bp)
- → TODO analyse results of gel
Plasmid Purification
Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids pSB1AC3 with medium and low constitutive promoters and GVP with the "Sygma-Aldrich™ 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
Gel
From each cup with isolated plasmid 5μL was mixed with 1μL 6x loading buffer and incubated for 10 minutes. Together with 1kB ladder all samples were loaded on a 1% agarose gel with 4μL EtBr.
Transporters
Metal Accumulation
MBP-ArsR fusion protein ligation was transformed for a second try, plated out on LB-amp100
(50 μL and concentrated after centrifugation)
Positive control (pSB1AC3-high constitutive promotor) ∞
Negative control (pSB1AC3-ccdb)1 colony on the concentrated plate
Ligations had no colonies.
Vectors
Dry