4 Matching Annotations
  1. Mar 2023
    1. Transcription of the araBAD genes under control of the pBAD promoter is induced about 300-fold above a basal uninduced level within 3 s of the addition of arabinose to cells growing on glycerol in minimal salts medium
    1. Wash the culture in fresh LB before adding glycerol, to avoid problems with reviving the culture later

      To prepare V. natriegens cells for −80 °C storage, an overnight culture of V. natriegens was washed in fresh medium before storage in glycerol. Cultures were centrifuged for 1 min at 20,000g and the supernatant was removed. The cell pellet was resuspended in fresh LB3 medium and glycerol was added to 20% final concentration. The stock was quickly vortexed and stored at −80 °C. Bacterial glycerol stocks stored in this manner are viable for at least five years.

      source: Lee, Henry H., et al. "Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi." Nature microbiology 4.7 (2019): 1105-1113.

  2. Dec 2019
    1. A single transformant may have a mutation at a low level that will eventually sweep through the population

      How does transforming a pure plasmid (miniprep) produce a mixed population?

      • Is a single colony on the transformation plate not really clonal due to evolution occurring in growth from the single founder cell?
      • And also the outgrowth steo prior to plating is introducing additional variation that differentiates different colonies
      • Hence adding these two statements together, picking 3 colonies from a plate streaked from a glycerol stock (derived from a clonal population) has lower variability than 3 colonies picked from a fresh transformation?

      Does this make any suggestion of using one or the other method for generating independent biological replicates?