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