3 Matching Annotations
  1. Nov 2020
    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. Jan 2020
    1. To prepare V. natriegens cells for −80 °C storage, an overnight culture of V. natriegens was washed in fresh medium before storage in glycerol
  3. 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?