Team:Amsterdam/Notebook/Protocols/Freeze thaw

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=Freeze/thaw cycle survival measurement=
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==Procedure==
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# Inoculate 3mL of LB from a glycerol stock<br>
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# Leave culture in 37°C stove for 2 hours<br>
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# Aliquot culture to 4 batchet of 500 μL<br>
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# Dillute 10μL of each aliquot 10.000x<br>Plate 100μL of resulting dilution on selective plate (Cycle = 0)<br>
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# Place remainder of aliquots in -20°C freezer for 2 hours<br>
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# Let aliquots thaw on ice for 1 hour<br>
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# Dillute 10μL of each aliquot 100x<br>Plate 100μL of resulting dilution on selective plate (Cycle = 1)<br>
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# Place remainder of aliquots in -20°C freezer for 2 hours<br>
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# Let aliquots thaw on ice for 1 hour<br>
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# Dillute 10μL of each aliquot 10x<br>Plate 100μL of resulting dilution on selective plate (Cycle = 2)<br>
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# Place remainder of aliquots in -20°C freezer for 2 hours<br>
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# Let aliquots thaw on ice for 1 hour<br>
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# Plate 100μL of remaining on selective plate (Cycle = 3)<br>
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# Count colony forming units (CFUs) on plates after ~15 hours
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<br>
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==Notes==
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* 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).
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* 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.
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{{:Team:Amsterdam/Footer}}

Revision as of 05:47, 21 September 2011

Freeze/thaw cycle survival measurement

Procedure

  1. Inoculate 3mL of LB from a glycerol stock
  2. Leave culture in 37°C stove for 2 hours
  3. Aliquot culture to 4 batchet of 500 μL
  4. Dillute 10μL of each aliquot 10.000x
    Plate 100μL of resulting dilution on selective plate (Cycle = 0)
  5. Place remainder of aliquots in -20°C freezer for 2 hours
  6. Let aliquots thaw on ice for 1 hour
  7. Dillute 10μL of each aliquot 100x
    Plate 100μL of resulting dilution on selective plate (Cycle = 1)
  8. Place remainder of aliquots in -20°C freezer for 2 hours
  9. Let aliquots thaw on ice for 1 hour
  10. Dillute 10μL of each aliquot 10x
    Plate 100μL of resulting dilution on selective plate (Cycle = 2)
  11. Place remainder of aliquots in -20°C freezer for 2 hours
  12. Let aliquots thaw on ice for 1 hour
  13. Plate 100μL of remaining on selective plate (Cycle = 3)
  14. 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.


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