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Team:Amsterdam/Notebook/Protocols/Freeze thaw
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# Leave culture in 37°C stove for 2 hours<br> | # Leave culture in 37°C stove for 2 hours<br> | ||
# Aliquot culture to 4 batchet of 500 μL<br> | # Aliquot culture to 4 batchet of 500 μL<br> | ||
| - | # | + | # Dilute 10μL of each aliquot 10.000x<br>Plate 100μL of resulting dilution on selective plate (Cycle = 0)<br> |
# Place remainder of aliquots in -20°C freezer for 2 hours<br> | # Place remainder of aliquots in -20°C freezer for 2 hours<br> | ||
# Let aliquots thaw on ice for 1 hour<br> | # Let aliquots thaw on ice for 1 hour<br> | ||
| - | # | + | # Dilute 10μL of each aliquot 100x<br>Plate 100μL of resulting dilution on selective plate (Cycle = 1)<br> |
# Place remainder of aliquots in -20°C freezer for 2 hours<br> | # Place remainder of aliquots in -20°C freezer for 2 hours<br> | ||
# Let aliquots thaw on ice for 1 hour<br> | # Let aliquots thaw on ice for 1 hour<br> | ||
| - | # | + | # Dilute 10μL of each aliquot 10x<br>Plate 100μL of resulting dilution on selective plate (Cycle = 2)<br> |
# Place remainder of aliquots in -20°C freezer for 2 hours<br> | # Place remainder of aliquots in -20°C freezer for 2 hours<br> | ||
# Let aliquots thaw on ice for 1 hour<br> | # Let aliquots thaw on ice for 1 hour<br> | ||
| Line 18: | Line 18: | ||
# Count colony forming units (CFUs) on plates after ~15 hours | # Count colony forming units (CFUs) on plates after ~15 hours | ||
<br> | <br> | ||
| + | |||
==Notes== | ==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). | * 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). | ||
Latest revision as of 06:25, 21 September 2011
Freeze/thaw cycle survival measurement
Procedure
- 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
- Dilute 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
- Dilute 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
- Dilute 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
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. Remember to make more preculture if you want to measure the OD in a 1mL cuvet with a fotospectrometer beforehand.
