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Wiki/Team:Warsaw/primers
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(New page: {{WarHead|headimg=2008.igem.org/wiki/images/b/b0/Logo_protocol.jpg}} <html> <h2>Primers</h2> <a name="Z_a_Z_o"><h3>Purification of His_Z_alpha and His_Z_omega</h3></a> <p>Culture ...)
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Revision as of 13:10, 26 September 2008
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PrimersPurification of His_Z_alpha and His_Z_omegaCulture E. coli producer strain in 3 ml of liquid LB medium + kanamycin for 8 hours. Then use it to inoculate 200 ml of liquid LB medium + kanamycin supplemented with 0,5 mM IPTG and grow it overnight. In the morning spin down the culture (5000 RPM, 10 min, 4 deg. C). Resuspend the pellet in PBS buffer and disrupt cells by sonication. Spin down sonication mixture (13200 RPM, 10 min, 4 deg. C) and discard supernatant – purified protein is present in sonication debris. Resuspend it in sterile ice cold ddH2O and store at 4 deg. C. Purification of His_A_alphaCulture, induce and disrupt E. coli in the same way as to purify His_Z_alpha. The protein is present in supernatant (about 10% of total protein) and can be added to selection medium without further purification. |
