Lidstrom:Preparing Freezer Cell Stocks: Difference between revisions
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*Centrifuge for ~2+ minutes & pour off supernatant. | *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. | *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. | *Store at -80oC. | ||
*Test stock after frozen a while. | *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." [http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/d8779pis.Par.0001.File.tmp/d8779pis.pdf Sigma/Source] | |||
==Glycerol== | ==Glycerol== | ||
* | * | ||
Latest revision as of 21:15, 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
