Lidstrom:Preparing Freezer Cell Stocks

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  • Grow ~ 5 mL of E. Coli overnight with appropriate antibiotics.
  • Centrifuge for ~2+ minutes & pour off supernatant.
  • Resuspend in 900 uL of HY (hypho) and 100 uL of filter-sterilized DMSO. Hypho is better than LB for this; I hear it buffers.
    • must use a filter that won't dissolve in DMSO . Most of the filters we have are not appropriate.
  • Store at -80oC.
  • Test stock after frozen a while.

Filtering DMSO: don't dissolve the filter

  • Pure DMSO dissolves many of the filters common in our lab.
  • "To prepare a sterile solution, use a teflon or nylon membrane to sterile-filter the DMSO; do not use a cellulose acetate membrane." Sigma/Source