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Revision as of 14:59, 9 September 2010

Expression Chaplins in NZ8900

-Grow O/N culture in Ty with antibiotics (Km5 and Cm5)

-Measure OD600 (with 0.9 ml with 100 μL of O/N culture, number *10=OD)

-Inoculate 15ml TY (with ab) in 100 ml flask to OD=0.1

-Grow till OD600 is approx. 0.5

-Take 2 ml t=0 sample

-Induce with 1% subtiline

-Continue growing

-Take 2 ml samples every hour

Chaplin purification

-5mg cell wall in 1.5 mL tube. Do not use gloves because static electric interaction

-Add app. 1 mL of TFA with a glass pasteur pipette.Do this in fumer and never leave TFA open for more than 30 sec!

-Vortex 1 min

-Spin down insoluble material for 10 min at 13000 rpm

-Transfer supernatant to new 1.5 mL eppendorf tube or 5 mL Greiner tube

-Dry TFA for 1-2 hours

-When tube is dry, resolve in water or Tris buffer