Team:University College London/Protocols/Competency3
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Revision as of 10:06, 2 August 2012
Generating Competency Day 2
Step 1 – Innoculation: Inoculate 100mls of Lysogeny Broth in pre-warmed conical with 1ml of the overnight culture from Day 2
Step 2 – Incubation: Incubate for two hours in a 37oC shaker until the cells are at the early log phase of the growth curve. Measure absorbance on a spectrometer until A6000 is approximately 0.3.
Step 3 – Incubation: Transfer to a chilled sterile 50ml Falcon tube and incubate on ice for 10 minutes Step 4 – Centrifuge:
-Time: 5 mins -G – 3300 -Temperature: 180C
Step 5 – Incubate: Resus in 10ml of ice cold 0.1M CaCl2 in 15% glycerol and incubate on ice for 30 minutes.
Step 6 – Centrifuge: -Time: 5 mins -G – 3300 -Temperature: 180C
Step 7 – Transfer: Resus in 1ml of ice cold 0.1M CaCl2 in 15% glycerol. Transfer 100ul aliquots to pre-chilled, pre-labelled eppendorf tubes. Store at -70oC
Step 8 – Centrifuge -Time: 5 mins -G – 3300 -Temperature: 180C
Step 9 - Storage: Store cells at -80oC