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Wiki/Team:Warsaw/protocols
From 2008.igem.org
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<a name="Z_a_Z_o"><h3>Purification of His_Z_alpha and His_Z_omega</h3></a> | <a name="Z_a_Z_o"><h3>Purification of His_Z_alpha and His_Z_omega</h3></a> | ||
| - | <p>Culture 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 | + | <p>Culture <i>E. coli</i> 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). | + 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). | ||
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<a name="A_a"><h3>Purification of His_A_alpha</h3></a> | <a name="A_a"><h3>Purification of His_A_alpha</h3></a> | ||
| - | <p>Culture, 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.</p> | + | <p>Culture, induce and disrupt <i>E. coli</i> 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.</p> |
</tr></table> | </tr></table> | ||
</html> | </html> | ||
Revision as of 15:43, 14 September 2008
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Culture 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. |
