Results of Chloramphenicol-100 Test
- Chloramphenicol plates at 100 µg/mL still did not work.
- Growth observed on both LB-only and LB-chlor(100) plates, for both DB3.1 and DH5alpha cells.
- These plates were made using the old batch of chloramphenicol that Calvin had made, not his newer batch.
- Will try spread plating Paul's batch of chloramphenicol.
Making Competent Cells
Checked the OD of the cells at 1220 (aim is to get A = [0.04,0.06])
- BW27781 A = 0.024
- DH5α A = 0.014
- Conclusion: Desired concentration not yet reached
- Put cells back into incubator
Checked the OD of the cells again at 1340
- BW27781 A = 0.084
- DH5α A = 0.024
- Conclusion: Concentration of BW27781 passed the desired amount. DH5α still not ready
- Put BW27781 into AMBL fridge to slow growth
- Put DH5α back into incubator
Checked OD of DH5α at 1430
- A = 0.112
- Conclusion: Concentration passed the desired amount.
- Put DH5α into AMBL fridge
- Since the concentrations of both strains have passed the accepted value, the experiment has been terminated.
- It was observed that many cuvettes are either dirty or scratched up. This will affect the accuracy of the OD reading. All cuvettes that are dirty or scratched up will be marked to prevent future inacurate reading.
- To increase the efficiency of making future competent cells, a growth curve will be created for each strain (BW27781, DH5α and DB3.1).
Creating Growth Curve
- Inoculated the 3 strains of cells (DB3.1, DH5α, BW27781) at 1520 in the Lagally Lab
- Used 50mL sterile LB + cells (frozen glycerol stock -80°C)
- Put into Lagally Lab shaker @90rpm and 37°C
- Paul's new batch of chloramphenicol from his lab.
- Freshly made chloramphenicol stocks.
- Made chloramphenicol stocks (1000X) at 25 mg/mL
- 250.2 mg of chloramphenicol
- 10 mL of MeOH (99.8% purity)
- Filter sterilized in 0.45 µm polyethersulfone sterile syringe filter from VWR.
- Stored in a sterile 15 mL culture tube
- Spread plated chloramphenicol stocks onto LB agar.
- One plate had final plated concentration of 25 µg/mL
- Another had a final plated concentration of 100 µg/mL
- Volumes plated are as follows:
||[chloramphenicol] stock (mg/mL)
||Vol. stock (µL)
||Vol MeOH (µL)
| Chlor 1
||Fresh 100% MeOH
| Chlor 2
||Fresh 100% MeOH
| Chlor 3
||Paul\'s Chloramphenicol stocks
- Streaked bacteria on plates
- Each plate was divided into two, with the DH5a cells (from glycerol stocks) streaked on one half and the DB3.1 cells (from glycerol stocks) streaked on the other half.
- Incubated at 37ºC in the Lagally Lab incubator at 6:16 PM.