Lidstrom:Preparing Freezer Cell Stocks

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(New page: Back to protocols Grow ~ 5 mL of E. Coli overnight with appropriate antibiotics. Centrifuge for 5-10 minutes & pour off supernatant. Resuspend in 900 uL of HY (hyp...)
Current revision (23:15, 6 June 2013) (view source)
 
(7 intermediate revisions not shown.)
Line 1: Line 1:
-
Back to [[Lidstrom: Protocols|protocols]]
+
Back to [[Lidstrom:Protocols|protocols]]
-
Grow ~ 5 mL of E. Coli overnight with appropriate antibiotics.
+
==DMSO==
-
Centrifuge for 5-10 minutes & pour off supernatant.
+
*Grow ~ 5 mL of E. Coli overnight with appropriate antibiotics.
-
Resuspend in 900 uL of HY (hypho) and 100 uL of filter-sterilized DMSO.
+
*Centrifuge for ~2+ minutes & pour off supernatant.
-
Store at -80oC.
+
*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.
+
** ''' 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." [http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/d8779pis.Par.0001.File.tmp/d8779pis.pdf Sigma/Source]
 +
 
 +
==Glycerol==
 +
*

Current revision

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

Personal tools