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Remove the glycerol stock from the -80 °C freezer. Work quickly so it does not thaw.
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Using a sterile pipet tip, scrape a small aliquot of the stock (>25 μl) into a sterile microfuge tube on ice. Replace the original stock at -80 °C and allow the scraping to thaw.
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Subculture 25 μl of the scraping into 2 ml LBS (1:80 subculture) and grow at 25 °C with aeration until the culture reaches an OD600 of approximately 0.3. Record the OD.
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Subculture 16.5 μl of the culture into 3 ml of the selective medium (1:181 subculture) and grow until the OD reaches the original OD. Note that 27.5 = 181, so the culture has now grown approximately 7.5 generations (doublings) in the selective medium. At this point freeze down an aliquot as a "frozen fossil record" and also proceed with the next step to grow the next 7.5 generations.
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Subculture 16.5 μl of the culture from above and proceed to grow as above for the next 7.5 generations in selective medium. Note: if inoculating from a glycerol stock, use 24.75 μl to account for the glycerol volume. Once the OD returns, freeze the glycerol stock as before.
Notes:
- If growing for essential genes and the selective medium is LBS, then note that the strains get an extra ~ 6 generations in the LBS pregrowth. We have typically kept this pregrowth the same.
- What if the growth rates differ? This has to be examined biologically and is best examined with relevant control conditions.