Difference between revisions of "Lidstrom:Preparing Freezer Cell Stocks"

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Back to [[Lidstrom:Protocols|protocols]]
 
Back to [[Lidstrom:Protocols|protocols]]
  
Grow ~ 5 mL of E. Coli overnight with appropriate antibiotics.
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==DMSO==
Centrifuge for 5-10 minutes & pour off supernatant.
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*Grow ~ 5 mL of E. Coli overnight with appropriate antibiotics.
Resuspend in 900 uL of HY (hypho) and 100 uL of filter-sterilized DMSO.
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*Centrifuge for ~2+ minutes & pour off supernatant.
Store at -80oC.
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*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.
Test stock after frozen a while.
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** ''' must use a filter that won't dissolve in DMSO '''.  Most of the filters we have are not appropriate.  
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*Store at -80oC.
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*Test stock after frozen a while.
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=== Filtering DMSO: don't dissolve the filter ===
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* Pure DMSO dissolves many of the filters common in our lab.
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* "To prepare a sterile solution, use a teflon or nylon membrane to sterile-filter the DMSO; do not use a cellulose acetate membrane." [http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/d8779pis.Par.0001.File.tmp/d8779pis.pdf Sigma/Source]
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==Glycerol==
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Latest revision as of 20:15, 6 June 2013

Back to protocols

DMSO

  • 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

Glycerol