Wiki/Team:Warsaw/primers

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(Difference between revisions)
(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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<h2>Primers</h2>
<h2>Primers</h2>
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<a name="Z_a_Z_o"><h3>Purification of His_Z_alpha and His_Z_omega</h3></a>
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<h3>One-armed bandit preparation</h3>
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<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
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<table style="font-size: 11px">
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<tr>
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<td>Primer name</td><td>Sequence</td>
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</tr>
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<tr>
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<td><a name="T7lLinkB">T7lLinkB </a>
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</td>
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<td>5' CGGATCCGGCGGTGGCGGTAGTGGAGGAGGTGGGTCTGGAGGCGGAGGGAACACGATTAACATCGCTAAGAAC  3'</td>
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</tr>
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+ 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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<tr>
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<td><a name="T7lRBSHi">T7lRBSHi</a>
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</td>
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<td>5' CACAAGCTTAGGAGGTGTACTAGATGAACACGATTAACATCGCTAAG  3'</td>
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</tr>
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Resuspend the pellet in PBS buffer and disrupt cells by sonication. Spin down sonication mixture (13200 RPM, 10 min, 4 deg. C) and discard
 
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supernatant – purified protein is present in sonication debris. Resuspend it in sterile ice cold ddH2O and store at 4 deg. C.</p>
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<tr>
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<td><a name="T7pXbSal">T7pXbSal</a>
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</td>
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<td>5' TCTGTCGACTCTAGATTACGCGAACGCGAAGTCC  3'</td>
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</tr>
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<tr>
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<td><a name="AIDlNrH">AIDlNrH</a>
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</td>
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<td>5' CGTAAGCTTAGGAGGTGTAGACCATGGACAGCCTCTTGATG  3'</td>
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</tr>
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<tr>
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<td><a name="AIDpLinB">AIDpLinB</a>
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</td>
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<td>5' CCACTACCGCCACCGCCGGATCCGCCACCTCCGCTGCCTCCACCACCAAGTCCCAAAGTACGAAATGC  3'</td>
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</tr>
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</table>
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<a name="A_a"><h3>Purification of His_A_alpha</h3></a>
 
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<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 13:20, 26 September 2008

Gallery Bricks Notebook Team Project Home

 

Primers

One-armed bandit preparation

Primer nameSequence
T7lLinkB 5' CGGATCCGGCGGTGGCGGTAGTGGAGGAGGTGGGTCTGGAGGCGGAGGGAACACGATTAACATCGCTAAGAAC 3'
T7lRBSHi 5' CACAAGCTTAGGAGGTGTACTAGATGAACACGATTAACATCGCTAAG 3'
T7pXbSal 5' TCTGTCGACTCTAGATTACGCGAACGCGAAGTCC 3'
AIDlNrH 5' CGTAAGCTTAGGAGGTGTAGACCATGGACAGCCTCTTGATG 3'
AIDpLinB 5' CCACTACCGCCACCGCCGGATCCGCCACCTCCGCTGCCTCCACCACCAAGTCCCAAAGTACGAAATGC 3'

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