Revision as of 05:49, 21 September 2011

Freeze/thaw cycle survival measurement

Inoculate 3mL of LB from a glycerol stock
Leave culture in 37°C stove for 2 hours
Aliquot culture to 4 batchet of 500 μL
Dillute 10μL of each aliquot 10.000x
Plate 100μL of resulting dilution on selective plate (Cycle = 0)
Place remainder of aliquots in -20°C freezer for 2 hours
Let aliquots thaw on ice for 1 hour
Dillute 10μL of each aliquot 100x
Plate 100μL of resulting dilution on selective plate (Cycle = 1)
Place remainder of aliquots in -20°C freezer for 2 hours
Let aliquots thaw on ice for 1 hour
Dillute 10μL of each aliquot 10x
Plate 100μL of resulting dilution on selective plate (Cycle = 2)
Place remainder of aliquots in -20°C freezer for 2 hours
Let aliquots thaw on ice for 1 hour
Plate 100μL of remaining on selective plate (Cycle = 3)
Count colony forming units (CFUs) on plates after ~15 hours

Dilution rates may be varied depending on 1) the concentration of the culture after 2 hours in the stove (an OD600 of ~0.03 works great for wildtype E. coli) and 2) the degree of cold resistance of the culture (if your culture is very resistant to freeze/thaw cycles, for example because it's expressing CspC ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K538004 BBa_K538004]), you might need to dilute 10x for the third cycle to keep the cells at a countable level).
The amount of preculture may be varied to allow more or less freeze/thaw cycles to be plated, but wildtype E. coli is unlikely to survive more than 3 cycles. Remember to make more preculture if you want to measure the OD in a 1mL cuvet with a fotospectrometer beforehand.

@@ Line 20: / Line 20: @@
 ==Notes==
 * Dilution rates may be varied depending on '''1)''' the concentration of the culture after 2 hours in the stove (an OD600 of ~0.03 works great for wildtype ''E. coli'') and '''2)''' the degree of cold resistance of the culture (if your culture is very resistant to freeze/thaw cycles, for example because it's expressing ''CspC'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K538004 BBa_K538004]), you might need to dilute 10x for the third cycle to keep the cells at a countable level).
-* The amount of preculture may be varied to allow more or less freeze/thaw cycles to be plated, but wildtype ''E. coli'' is unlikely to survive more than 3 cycles.
+* The amount of preculture may be varied to allow more or less freeze/thaw cycles to be plated, but wildtype ''E. coli'' is unlikely to survive more than 3 cycles. Remember to make more preculture if you want to measure the OD in a 1mL cuvet with a fotospectrometer beforehand.
 {{:Team:Amsterdam/Footer}}