50 Life Technologies
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| Selection and Stable Pool Considerations
Develop a naming convention for pools before starting selection to
avoid confusion as to which flask has which concentration of selection
reagents; for example:
• For Phase I, use "10" to represent 10P/100M (10 µg/mL
puromycin, 100 nM methotrexate), and "20" to represent
20P/200M.
• For Phase II, use "30" to represent 30P/500M, and "50" to
represent 50P/1,000M.
– For example, pool 10–30 received low selection pressure
in both selection phases.
Prepare media containing puromycin and methotrexate fresh each time.
• Puromycin and methotrexate can be added to each flask
individually, or, when multiple flasks require the same selection
pressure, to a larger volume of medium to be used to passage
multiple flasks.
Prewarm selection medium (complete CD FortiCHO
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Medium
supplemented with Anti-Clumping Agent at 1:100 dilution) to 37°C.
Make sure puromycin is in solution before opening a freshly thawed tube.
• Puromycin can form a white powder at the bottom of the tube
when thawing. Ensure that the drug is in solution by inverting the
tube several times before opening.
Selection and Stable Pool Considerations
Do:
