IGEM:IMPERIAL/2006/LabCalendar/2006-7-10
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John Sy
Culturing Bacteria
- Combine together:
- 10 mL growth medium
- 200 uL DH5a E. coli gram negative bacteria
- Allow to grow for a few hours until the optical density of the bacterial solution is between 0.8 and 1.0 as measured by the spectrophotometer
- Cool the culture for a few minutes and place in an ice bath
- Centrifuge the bacteria to obtain a bacterial pellet
- Remove media and place on ice
- Add 1 mL of 100 mM calcium chloride solution
- Vortex and place back on ice bath
- Transfer 1 mL into a microcentrifuge tube and place back on ice
- Remove supernatant and resuspend in 100 uL of calcium chloride
Innoculation
- Innoculate a colony of bacteria into 2 mL of ampicillin resistant growth medium
Transfering the part from the registry into the bacteria
- We use part I13273 from well 4H (YFP Producer controleld by 3OC6HSL Receiver Device (LuxR based receiver controls production of YFP)
- Pipette 15 uL of ultrapure water into the well and dilute
- Remove and transfer into a microvial
- Remove 5 uL of DNA solution and place into solution containing the bacteria
- Leave solution for about 40 minutes
- Heat shock the soution at 42C for 90 sec and place back on ice bath
- Transfer the bacteria with the part onto an agar plate and spread out using a glass spreader
- Incubate at 37C overnight
