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Team:Groningen/Chaplins
From 2010.igem.org
(New page: '''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 (wi...) |
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-When tube is dry, resolve in water or Tris buffer | -When tube is dry, resolve in water or Tris buffer | ||
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| + | '''Sample treatment''' | ||
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| + | -Take 2ml sample and spin down | ||
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| + | '''Supernatant''' | ||
| + | |||
| + | -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 | ||
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| + | '''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) | ||
| + | |||
| + | -Speedvacum | ||
| + | |||
| + | -TFA treatment | ||
Latest revision as of 17:02, 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
Sample treatment
-Take 2ml sample and spin down
Supernatant
-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)
-Speedvacum
-TFA treatment
