Revision as of 17:02, 9 September 2010 by Neima (Talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

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

Sample treatment

-Take 2ml sample and spin down


-1.5 ml of supernant in separate cup, rest of supernatant can be discarted (at this point you can store in freezer)

-TCA precipitation until drying pellet after washing with aceton

-Continue with TFA treatment

Cell pellet

-TFA treatment

-Pellet of TFA treatment with cells should be airdried

-Lysed for half and out at 37 °C (P-buffer with 20 mg/ml lysozyme)


-TFA treatment