This protocol outlines the steps to grow up your libraries and perform chemical genomics for sequence quantification.
- Library
- Culture tubes
- Eppitubes
- LB (Luria-Bertani) media
- 250 mL flasks
- PCR tubes
- Falcon tubes
- Assign Alphanumeric Labels: Assign alphanumeric labels for each sample and print out a "cheat sheet."
- Calculate Dilutions for IPTG:
- Volume of IPTG to add = (Final Volume of Culture / 1000)
- Note: The dilution factor for IPTG is 1000 (from 1M to 1mM).
- Prep 250 mL Flasks: Prepare 250 mL flasks for T0 collections.
- Label Eppitubes: Prep Eppitubes with labels for pellet collection.
- Inoculate Flask: In a 250 mL flask, add 35 mL LB. To achieve a starting OD of 0.02, use the following volumes based on the stock OD:
- For E. coli with stock OD = 25: Use 28 uL
- For E. cloacae with stock OD = 30: Use 23 uL
- For K. pneumoniae with stock OD = 30: Use 23 uL
- Equation: Volume to inoculate = (Desired OD x Culture Volume) / Stock OD
- Incubate: Place the flask in a 37°C shaker until the OD reaches 0.2.
- Collect T0 Samples: Take 10 mL from each flask and distribute into Falcon tubes for each of the 3 replicates, including a backup for each. Note: 10 mL at OD 0.2 is approximately equivalent to 1 mL at OD 2.
- Prep for DNA Extraction: Utilize the entire pellet for DNA extraction.
- Prep Overnight Tubes for T1: Inoculate another overnight tube with 1 mL from the Falcon tube to achieve a starting OD of 0.02.
- Incubate Tubes: Place tubes in a tube rack and incubate in a 37°C shaker for 18 hours.
- Measure OD: After 18 hours of incubation, extract 100 uL from each T1 tube. Dilute this into 900 uL of LB for a 1:10 dilution and measure the OD.
- Prep for DNA Extraction: Take 1 mL, spin down, and store for DNA extraction. Take an additional 1 mL as a backup.
- Prep for Next Timepoint (Tn): Inoculate another overnight tube with the aim of reaching an OD of 0.02 in 4 mL. Use 40 uL from the previous overnight.
- Incubate Tubes: Place tubes in a 37°C shaker for 18 hours.
Repeat steps 1-4 for each subsequent day until you are done with the experiment.
❌
Attempt 2 - September 17-19, 2023 (E. coli induction only)Failure: Sulfur smell and unequal ODs
Note: The aim is to inoculate cultures at an OD600 of 0.02.
Day 1 - T0 Prep (September 17, 2023)
T0 In and T0 Out OD600 Measurements
Dilution and Induction (September 18, 2023)
T1 In and T1 Out OD600 Measurements
T2 In and T2 Out (September 18-19, 2023)
T2 In and T2 Out OD600 Measurements
T3 In and T3 Out (September 19, 2023)
Note: Tube 3F had a sulfur-like smell.
T3 In and T3 Out OD600 Measurements