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 1 - September 14-16, 2023 (E. coli induction only)Failure: Inoculated at too low of a dilution (4uL instead of 40uL during passages)
September 15, 2023 - 2:10 PM (T1)
September 16, 2023 - 10:10 AM (T2)
Here, I realized I was inoculating at too low of an inoculum. I was diluting 1:1000, with 4uL.
Redo with appropriate dilution of 1:100, with 40 uL.