IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/06/01

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Results of Chloramphenicol Testing

 * Chloramphenicol plates did not work.
 * Growth was observed on both the LB-only and LB+Chloramphenicol plates.
 * Both DB3.1 and DH5alpha cells grew on both sets of plates.
 * Could possibly be an issue with the concentration. See here.
 * This page states that chloramphenicol plates should be made at 100 µg/mL concentration rather than 25 µg/mL.
 * To do this, we would need to plate 80 µL on each LB plate rather than 20 µL.
 * New plates will be made in the afternoon.

Testing of Chloramphenicol at 100 µg/mL Concentration

 * Used two LB-agar plates
 * One for LB-only control
 * One for LB + Chlor-100 test
 * Plated 80 µL of 25 mg/mL chloramphenicol onto 20 mL LB agar plate for a final concentration of 100 µg/mL
 * Let dry for 1 hour.
 * Plates were divided in two, and both DH5alpha and DB3.1 were streaked, each on one half of the plate.
 * The plates were then incubated in the Lagally Lab 37ºC incubator at 1:32PM ON.
 * Will check them tomorrow to see if the higher concentration worked.

Results of Test Plasmid pNHG1 with DH5α and DB3.1

 * DB3.1 and DH5α both grew on Tet and Kan plates
 * Both cells were able to pick up a resistant plasmid and express the resistance gene
 * Indicates that the failure of the Tet Construction plasmid and Kan Construction plasmid may be caused by the loss of function in the plasmid

Results of 09/05/29 DH5α and BW27783 Competency Test

 * Neither DH5α or BW27783 showed any growth when plated onto an Amp plate
 * Indicates that the cells did not become competent
 * Will transform both strains again with pUC19 (Amp resistance) to make sure of the result
 * 5mL of DH5 and 5mL of BW27783 was innoculated and grown overnight in a 30°C incubator to make new competent cells tomorrow if a test cells do not grow

Transformation of DH5α and BW27783

 * Thaw 100uL of DH5α and BW27783 glycerol stocks on ice
 * Add 2uL of pUC19 plasmid to each of the stocks
 * Keep on ice for 30min
 * Heat shock at 42°C for 60 seconds
 * Keep on ice for 3 minutes
 * Add 400uL of LB, the LB added to the BW27783 cells was contaminated
 * Recover at 37°C for 2 hours
 * Spread plate cells on Amp plates


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