IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/06/03

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Chloramphenicol Testing Results

  • This is a continuation of previous day's test.
  • Took plates out at 10:15 am.
  • Results are as follows:
Plate Growth for DB3.1 Growth for DH5a
Chlor 1 Yes Yes
Chlor 2 Yes Yes
Chlor 3 No No
  • Conclusions:
    • Our batch of chloramphenicol must be faulty, or we are preparing it the wrong way. It may also be the wrong form of Chlor.
    • For now, I will call Paul to see if we can borrow his lab's chloramphenicol.
    • Plates were left on Roza's bench at 10:26 a.m.

Preparation of LB-Agar Plates

  • In anticipation of the need for more LB-Agar, I will make more LB-Agar and ask someone to pour the plates this afternoon when it is autoclaved.
  • LB Agar composition was:
    • 13g of LB-Agar in 350 mL of sdH2O.
    • Prepared two bottles.

Chloramphenicol Testing for Paul's new batch

  • Paul brought over a new batch of chloramphenicol from his lab for us to use.
  • Stock was made at 25 mg/mL in 100% MeOH.
  • Steps taken to test chloramphenicol:
    • Spread plated 25 µL of new chloramphenicol onto 1 LB agar plate (Chlor 1). LB-only is the control (Chlor 0)
    • Let dry for 10 minutes.
    • Streaked DB3.1 and DH5a onto each of the plates as done previously.
    • Stored at Lagally Lab 37ºC incubator at 11:43 AM.
    • Paul's chloramphenicol stored at Lagally Lab -20ºC freezer; will make aliquots of them after confirmation of efficacy.

Growth Curve for E. coli DB3.1, DH5a and BW27783

  • The purpose of doing this is to take the guesswork out of growing up the cells for competency. Frequently, the cells have been grown at 30ºC for just too long such that the OD >0.6, which is the max OD that can be tolerated for making competent cells.

Steps Taken

  • AMBL shaking incubator was pre-warmed to 30ºC, the temperature at which competent cells have to be pre-grown at.
  • HyClone DPBS/Modified (-Calcium, -Magnesium) from Thermo Scientific was pre-chilled in ice water bath.
  • Prepared test tubes with 9 mL or 9.9 mL of LB (18 tubes each)
  • Prepared two 96-well dilution plates with either 900µL or 990µL/well for dilutions
  • Measured OD of each sample
Strain Dilution OD600 Time Measured Actual OD660 Bacterial V1 (mL) To-Up
BW27783 1 in 20 0.663 2:50 PM 13.26 0.49 49.5
DB3.1 1 in 20 0.384 2:52 PM 7.68 0.85 49.2
DH5a 1 in 10 0.801 2:53 PM 8.01 0.81 49.2
  • Put 50 mL of bacteria in each flask. Remeasured OD600:
Strain OD600 Time Measured
BW27783 0.103 3:09 PM
DB3.1 0.133 3:09 PM
DH5a 0.107 3:09 PM

Results for OD Readings

Time Strain OD600 (1) OD600 (2)
3:44 PM BW27783 0.014 0.016
3:46 PM DH5a 0.016 0.008
3:51 PM DB3.1 0.092 0.086
4:18 PM BW27783 0.085 0.084
4:21 PM DH5a 0.135 0.149
4:22 PM DB3.1 0.223 0.228
4:42 PM BW27783 0.078 0.073
4:43 PM DH5a 0.117 0.111
4:44 PM DB3.1 0.259 0.244
5:08 PM BW27783 0.070 0.075
5:09 PM DH5a 0.095 0.095
5:10 PM DB3.1 0.355 0.354

Comments on Results

  1. OD readings fluctuated throughout the timecourse, and this was not as expected.
  2. When Calvin did his OD readings, he noticed that the blank (LB-only) read 0.2 when measured as a "sample" after measuring the prior samples.
  3. This Friday, we will repeat the experiment with the following modifications:
    1. No plating of bacteria - because we are not interested in the number of cells (too troublesome)
    2. We will be using the Finlay Lab spectrophotometer to do the OD measurements.
    3. The experiment will be started earlier at 9:00 AM to finish off earlier.
  4. The plates that were plated today are placed into the AMBL 37ºC incubator at 6:30PM. They will be checked tomorrow.

Summary of the Day

  1. First batch of chloramphenicol from Paul's lab worked while ours didn't.
  2. Paul made a new batch of chloramphenicol and they are being tested now.
  3. Growth curve for E. coli DB3.1, DH5a and BW27783 did not work as expected.
  4. (Not recorded) Poured more LB-agar with Janny. All stored in AMBL 4ºC.