- Mar 2023
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www-nature-com.ezproxy.rice.edu www-nature-com.ezproxy.rice.edu
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To prepare V. natriegens cells for −80 °C storage, an overnight culture of V. natriegens was washed in fresh medium before storage in glycerol
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barricklab.org barricklab.org
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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.
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- Dec 2019
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jbioleng.biomedcentral.com jbioleng.biomedcentral.com
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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?
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