Difference between revisions of "Team:Bielefeld-CeBiTec/Notebook/Mutation"

 
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<body>
 
<body>
 
<div class="container main">
 
<div class="container main">
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<div class="container text_header"><h1>lab notebook.</h1></div>
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<div class="jumbotron" style="background-image: url(https://static.igem.org/mediawiki/2016/a/ab/Bielefeld_CeBiTec_2016_10_14_X_notebook.png)">
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<div class="container text_header"><h3>Mutation</h3></div>
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<div class="jumbotron-text">
 +
<h1 style="margin-bottom: 0px; text-align:left">Lab Notebook</h1>
 +
<h2 style="color:#ffffff; text-align:left">Documenting the Mutation</h2>
 +
</div>
 +
</div>
 +
<p style="position: relative; font-size: 9px; top: -50px; color: white; float: right; right: 10px;"></p>
 +
 
 +
<div class="container text_header"><h3>Mutation</h3></div><br>
 
<div class=”container text” align="justify">
 
<div class=”container text” align="justify">
  
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<button data-toggle="collapse" data-target="#1M">Week 1: (04.04.16-10.04.16):</button>  
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<button data-toggle="collapse" class="btn-block"  data-target="#1M">Week 1: (04.04.16-10.04.16):</button>  
 
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<div id="1M" class="collapse">  
 
<br>
 
<br>
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<br>
 
<br>
  
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<button data-toggle="collapse" data-target="#2">Week 2: (11.04.16-17.04.16): </button>  
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<button data-toggle="collapse" class="btn-block"  data-target="#2">Week 2: (11.04.16-17.04.16): </button>  
 
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<div id="2" class="collapse">  
 
<br>
 
<br>
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<br>
 
<br>
  
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<button data-toggle="collapse" data-target="#3">Week 3: (18.04.16-24.04.16): </button>  
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<button data-toggle="collapse" class="btn-block"  data-target="#3">Week 3: (18.04.16-24.04.16): </button>  
 
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<br>  
 
<br>  
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<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#4">Week 4: (25.04.16-01.05.16): </button>  
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<button data-toggle="collapse" class="btn-block"  data-target="#4">Week 4: (25.04.16-01.05.16): </button>  
 
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<br>  
 
<br>  
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<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#5">Week 5: (02.05.16-08.05.16): </button>  
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<button data-toggle="collapse" class="btn-block"  data-target="#5">Week 5: (02.05.16-08.05.16): </button>  
 
<div id="5" class="collapse">  
 
<div id="5" class="collapse">  
 
<br>  
 
<br>  
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<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#6">Week 6: (09.05.16-15.05.16): </button>  
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<button data-toggle="collapse" class="btn-block"  data-target="#6">Week 6: (09.05.16-15.05.16): </button>  
 
<div id="6" class="collapse">  
 
<div id="6" class="collapse">  
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li>Transformation in DH5&alpha;:</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;:</li>
 
<ul>
 
<ul>
 
<li>pHSG-WTPolI</li>
 
<li>pHSG-WTPolI</li>
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<li>pSBIA3:E0044</li>
 
<li>pSBIA3:E0044</li>
 
</ul>
 
</ul>
−
<ul>
 
 
<li>Everything besides pSBIA3:E0044 grew. Taking overnight cultures at 37&thinsp;&deg;C.</li>
 
<li>Everything besides pSBIA3:E0044 grew. Taking overnight cultures at 37&thinsp;&deg;C.</li>
 
<li>Plating provided JS200 strain out.</li>
 
<li>Plating provided JS200 strain out.</li>
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<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#7">Week 7: (16.05.16-22.05.16): </button>  
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<button data-toggle="collapse" class="btn-block"  data-target="#7">Week 7: (16.05.16-22.05.16): </button>  
 
<div id="7" class="collapse">  
 
<div id="7" class="collapse">  
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li>Plasmid isolation of: </li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
 +
of: </li>
 
<ul>
 
<ul>
 
<li>pHSG-WTPolI</li>
 
<li>pHSG-WTPolI</li>
 
<li>pHSG-EPPolI</li>
 
<li>pHSG-EPPolI</li>
 
<li>pLA230</li>
 
<li>pLA230</li>
−
<li>psBIK3:J04450</li>
+
<li>pSBIK3:J04450</li>
 
</ul>
 
</ul>
 
<li>Overnight cultures from JS200. </li>
 
<li>Overnight cultures from JS200. </li>
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<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#8">Week 8: (23.05.16-29.05.16): </button>  
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<button data-toggle="collapse" class="btn-block"  data-target="#8">Week 8: (23.05.16-29.05.16): </button>  
 
<div id="8" class="collapse">  
 
<div id="8" class="collapse">  
 
<br>
 
<br>
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<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#9">Week 9: (30.05.16-05.06.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#9">Week 9: (30.05.16-05.06.16): </button>  
 
<div id="9" class="collapse">  
 
<div id="9" class="collapse">  
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li>Transformation in DH5&alpha;:</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;:</li>
 
<ul>
 
<ul>
 
<li>pSBIC3:E1010</li>
 
<li>pSBIC3:E1010</li>
 
<li>pSBIC3:K731722</li>
 
<li>pSBIC3:K731722</li>
 
</ul>
 
</ul>
−
<li>Transformation in JS200;:</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in JS200;:</li>
 
<ul>
 
<ul>
 
<li>pHSG-EPPolI & pLA230</li>
 
<li>pHSG-EPPolI & pLA230</li>
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−
<button data-toggle="collapse" data-target="#10">Week 10: (06.06.16-12.06.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#10">Week 10: (06.06.16-12.06.16): </button>  
 
<div id="10" class="collapse">  
 
<div id="10" class="collapse">  
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li> Overnight cultures of transformation from week 9.</li>
+
<li> Overnight cultures of <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">transformation</a>  from week 9.</li>
−
<li>Plasmid isolation & restriction with Spe&thinsp;I. </li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
 +
& <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">restriction digestion</a>
 +
with Spe&thinsp;I.</li>
 
<li>Gel</li>
 
<li>Gel</li>
 
<li>Idea: BioBrick site directly behind pMBi ori of pSBIK3</li>
 
<li>Idea: BioBrick site directly behind pMBi ori of pSBIK3</li>
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<li>1. Deletion of BioBrick site</li>
 
<li>1. Deletion of BioBrick site</li>
 
<ul>
 
<ul>
−
<li>Q5 PCR</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
</li>
 
<ul>
 
<ul>
 
<li>Template: pSBIK3:J04450</li>
 
<li>Template: pSBIK3:J04450</li>
−
<li>Part 1 with primer CH07/CH21, temp=56&deg;C</li>
+
<li>Part 1 with primer pSB1K3_Del_for
−
<li>Part 2 with primer CH08/CH20, temp=57&deg;C</li>
+
/pSB1K3_D_for_P2
 +
, temp = 56&thinsp;&deg;C</li>
 +
<li>Part 2 with primer pSB1K3_Del_rev
 +
/pSB1K3_D_rev_P1
 +
, temp = 57&thinsp;&deg;C</li>
 
<li>-> Part 1 worked; part 2 not</li>
 
<li>-> Part 1 worked; part 2 not</li>
 
</ul>
 
</ul>
−
<li>Transformation in DH5&alpha; of pSBIC3:K592100</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha; of pSBIC3:K592100</li>
 
</ul>
 
</ul>
 
<li>2.: New BioBrick site behind ori</li>
 
<li>2.: New BioBrick site behind ori</li>
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<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#11">Week 11: (13.06.16-19.06.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#11">Week 11: (13.06.16-19.06.16): </button>  
 
<div id="11" class="collapse">  
 
<div id="11" class="collapse">  
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li>Restriction of pSBIK3:J04450 with Spe&thinsp;I and Xba&thinsp;I</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
−
<li>Colony PCR of K592100</li>
+
of pSBIK3:J04450 with Spe&thinsp;I and Xba&thinsp;I</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of K592100</li>
 
<li>Gradient PCR of part 1 & part 2</li>
 
<li>Gradient PCR of part 1 & part 2</li>
 
</ul>
 
</ul>
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<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#12">Week 12 (20.06.16-26.06.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#12">Week 12 (20.06.16-26.06.16): </button>  
 
<div id="12" class="collapse">  
 
<div id="12" class="collapse">  
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li>Q5 PCR at 67&thinsp;&deg;C:</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
at 67&thinsp;&deg;C:</li>
 
<ul>
 
<ul>
 
<li>template: pSBIK3:J04450; primer PS02/PS03</li>
 
<li>template: pSBIK3:J04450; primer PS02/PS03</li>
Line 154: Line 173:
 
<li>template: pSBIC3:K592100; primer PS00/PS01</li>
 
<li>template: pSBIC3:K592100; primer PS00/PS01</li>
 
</ul>
 
</ul>
−
<li>Gibson-Assembly: 15 &mu;l Gibson Mastermix + 3.5 &mu;l pSBIK3 Backbone + 1.5 &mu;l BFP-Insert</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a>: 15 &mu;l Gibson Mastermix + 3.5 &mu;l pSBIK3 Backbone + 1.5 &mu;l BFP-Insert</li>
−
<li>Transformation in DH5&alpha;</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;</li>
 
<li>12 clones on LB-Kan plate that glow blue!</li>
 
<li>12 clones on LB-Kan plate that glow blue!</li>
 
</ul>
 
</ul>
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<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#13">Week 13 (27.06.16-03.07.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#13">Week 13 (27.06.16-03.07.16): </button>  
 
<div id="13" class="collapse">  
 
<div id="13" class="collapse">  
 
<br>  
 
<br>  
 
<ul>
 
<ul>
 
<li>Gene synthesis is there (AraProm, dnaQ, dam-seqA, emrR, ugi-cda1)! Resolve in TE-buffer</li>
 
<li>Gene synthesis is there (AraProm, dnaQ, dam-seqA, emrR, ugi-cda1)! Resolve in TE-buffer</li>
−
<li>Gibson assembly of the gene synthesis in pSBIC3 backbone</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of the gene synthesis in pSBIC3 backbone</li>
−
<li>Transformation in NEB electro competent cells</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in NEB electro competent cells</li>
−
<li>Colony PCR with VR/VF</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li>
 
<li>Overnight culture from AraProm, dam-seqA, emrR</li>
 
<li>Overnight culture from AraProm, dam-seqA, emrR</li>
 
</ul>
 
</ul>
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<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#14">Week 14: (04.07.16-10.07.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#14">Week 14: (04.07.16-10.07.16): </button>  
 
<div id="14" class="collapse">  
 
<div id="14" class="collapse">  
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li>Plasmid isolation of the overnight cultures</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
−
<li>Restriction with EcoR&thinsp;I/ Pst&thinsp;I</li>
+
of the overnight cultures</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
with EcoR&thinsp;I/ Pst&thinsp;I</li>
 
<li>Sequencing of AraProm, dam-seqA, emrR</li>
 
<li>Sequencing of AraProm, dam-seqA, emrR</li>
−
<li>Transformation in DH5&alpha; of pSBIC3:K516132</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha; of pSBIC3:K516132</li>
−
<li>Repeated colony PCR of dnaQ, ugi-cda1 -> only empty plasmids -> new primer</li>
+
<li>Repeated <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of dnaQ, ugi-cda1 -> only empty plasmids -> new primer</li>
 
<li>overnight culture of pSBIC3:K516132</li>
 
<li>overnight culture of pSBIC3:K516132</li>
−
<li>Plasmid isolation of pSBIC3:K516132</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
−
<li>Q5 PCR with template pSBIC3:K516132, primer CH30/CH31, temp = 67 &deg;C</li>
+
of pSBIC3:K516132</li>
−
<li>DpnI digestion of pcr product pSBIC3:K516132, 1h 37&thinsp;&deg;C</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
with template pSBIC3:K516132, primer CH30/CH31, temp = 67 &deg;C</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
 +
of pcr product pSBIC3:K516132, 1h 37&thinsp;&deg;C</li>
 
<li>1% Agarosegel & purification of the 2&thinsp;kb band -> new backbone</li>
 
<li>1% Agarosegel & purification of the 2&thinsp;kb band -> new backbone</li>
−
<li>repeated Gibson assembly with the new backbone and ugi-cda1/ dnaQ</li>
+
<li>repeated <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> with the new backbone and ugi-cda1/ dnaQ</li>
−
<li>Transformation in DH5&alpha;</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;</li>
 
<ul>
 
<ul>
 
<li>pSBIA3:J04450</li>
 
<li>pSBIA3:J04450</li>
 
<li>pSBIC3:J06702</li>
 
<li>pSBIC3:J06702</li>
−
<li>GFP cd</li>
+
<li>GFP-cd</li>
 
<li>pSBIC:E2050</li>
 
<li>pSBIC:E2050</li>
 
<li>dnaQ Gibson</li>
 
<li>dnaQ Gibson</li>
 
<li>ugi-cda1 Gibson</li>
 
<li>ugi-cda1 Gibson</li>
 
</ul>
 
</ul>
−
<li>Overnight culture of DH5&alpha; with pSBIK3:RFPcd/ pSBIK3:BFPcd </li>
+
<li>Overnight culture of DH5&alpha; with pSBIK3:RFP-cd/ pSBIK3:BFP-cd </li>
 
<li>Testing of the fluorescent proteins RFP and BFP with the Teacan</li>
 
<li>Testing of the fluorescent proteins RFP and BFP with the Teacan</li>
−
<li>Colony PCR of pSBIC3:dnaQ with VR/VF</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of pSBIC3:dnaQ with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li>
 
<li>Overnight culture of good clones, pGFPuv, pSBIA3:J04450, pSBIC3:J06702</li>
 
<li>Overnight culture of good clones, pGFPuv, pSBIA3:J04450, pSBIC3:J06702</li>
 
</ul>
 
</ul>
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<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#15">Week 15: (11.07.16-17.07.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#15">Week 15: (11.07.16-17.07.16): </button>  
 
<div id="15" class="collapse">  
 
<div id="15" class="collapse">  
 
<br>
 
<br>
 
<ul>
 
<ul>
 
<li>Primer design for genesynthesis</li>
 
<li>Primer design for genesynthesis</li>
−
<li>Plasmid isolation of the overnight cultures</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
−
<li>Restriction of pSBIC3:dnaQ, mCherry, E2050-cds</li>
+
of the overnight cultures</li>
−
<li>Sequencing of dnaQ clones with VR/VF</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
−
<li>Colony-PCR with CH34/VR on ugi-cda1</li>
+
of pSBIC3:dnaQ, mCherry, E2050-cds</li>
 +
<li>Sequencing of dnaQ clones with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
with CH34/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
on ugi-cda1</li>
 
<li>Sequencing of ugi-cda1</li>
 
<li>Sequencing of ugi-cda1</li>
 
<li>Assembly of dnaQ, dam, seqA</li>
 
<li>Assembly of dnaQ, dam, seqA</li>
 
<ul>
 
<ul>
−
<li>Q5 PCR</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
</li>
 
<ul>
 
<ul>
−
<li>template pSBIC3:dnaQ, primer CH35/CH36, temp = 59.9&thinsp;&deg;C</li>
+
<li>template pSBIC3:dnaQ, primer dnaQ_Gib_for
−
<li>template pSBIC3:dam-seqA, primer CH37/CH38, temp = 58.3&thinsp;&deg;C</li>
+
/dnaQ_Gib_rev
 +
, temp = 59.9&thinsp;&deg;C</li>
 +
<li>template pSBIC3:dam-seqA, primer dam_Gib_for
 +
/dam_Gib_rev
 +
, temp = 58.3&thinsp;&deg;C</li>
 
<li>template pSBIC3:dam-seqA, primer CH39/CH40, temp = 57&thinsp;&deg;</li>
 
<li>template pSBIC3:dam-seqA, primer CH39/CH40, temp = 57&thinsp;&deg;</li>
−
<li>template pHSG-WTPolI, primer CH42/CH43, temp = 58.4&thinsp;&deg;C</li>
+
<li>template pHSG-WTPolI, primer PolI_Gib_for
−
<li>template pHSG-EPPolI, primer CH42/CH43, temp = 58.4&thinsp;&deg;C</li>
+
/PolI_Gib_rev
 +
, temp = 58.4&thinsp;&deg;C</li>
 +
<li>template pHSG-EPPolI, primer PolI_Gib_for
 +
/PolI_Gib_rev
 +
, temp = 58.4&thinsp;&deg;C</li>
 
</ul>
 
</ul>
−
<li>DpnI digestion, gel extraction</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
−
<li>Restriction of pSBIC3:RFPcd with Xba&thinsp;I/Spe&thinsp;I, gel extraction of 2&thinsp;kb band</li>
+
, <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of pSBIC3:RFP-cd with Xba&thinsp;I/Spe&thinsp;I, <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">gel extraction</a>
 +
of 2&thinsp;kb band</li>
 
</ul>
 
</ul>
 
</ul>
 
</ul>
Line 233: Line 277:
 
<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#16">Week 16: (18.07.16-24.07.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#16">Week 16: (18.07.16-24.07.16): </button>  
 
<div id="16" class="collapse">  
 
<div id="16" class="collapse">  
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li>Gibson of dnaQ, dam, seqA, C3 backbone</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of dnaQ, dam, seqA, C3 backbone</li>
−
<li>Transformation in KRX compis</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in KRX compis</li>
−
<li>Colony-PCR with the Gibson clones -> no bands</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
−
<li>Q5 PCR</li>
+
with the Gibson clones -> no bands</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
</li>
 
<ul>
 
<ul>
 
<li>template pSBIC3:dnaQ, primer CH35/36, temp = 59.9&thinsp;&deg;C</li>
 
<li>template pSBIC3:dnaQ, primer CH35/36, temp = 59.9&thinsp;&deg;C</li>
Line 247: Line 293:
 
<li> template pSBIK3:J04450, primer CH30/31, temp = 67&thinsp;&deg;C </li>
 
<li> template pSBIK3:J04450, primer CH30/31, temp = 67&thinsp;&deg;C </li>
 
</ul>
 
</ul>
−
<li>DpnI distriction and gel extraction of pSBIK3 backbone</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
−
<li>Gibson assembly of dnaQ, dam, seqA in K3 backbone (ASI)</li>
+
and <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">gel extraction</a>
−
<li>Transformation in DH5&alpha;</li>
+
of pSBIK3 backbone</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of dnaQ, dam, seqA in K3 backbone (ASI)</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
 
<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#17">Week 17: (25.07.16-31.07.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#17">Week 17: (25.07.16-31.07.16): </button>  
 
<div id="17" class="collapse">  
 
<div id="17" class="collapse">  
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li>Solony-PCR of ASI with VR/VF -> plating positive clones out</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of ASI with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a> -> plating positive clones out</li>
 
<li>Sequencing of positive clones</li>
 
<li>Sequencing of positive clones</li>
−
<li>Q5 PCR</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
</li>
 
<ul>
 
<ul>
 
<li>template pSBIC3:emrR, primer BF1/2</li>
 
<li>template pSBIC3:emrR, primer BF1/2</li>
Line 268: Line 319:
 
<li> template pHSG:WTPolI, primer CH42/43</li>
 
<li> template pHSG:WTPolI, primer CH42/43</li>
 
</ul>
 
</ul>
−
<li>PCR clean up of the Q5 PCR</li>
+
<li>PCR clean up of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
−
<li>Gibson assembly of emrR, ugi, cda1 in pSBIK3 backbone (ASII)</li>
+
</li>
−
<li>Gibson assembly of EPPolI/ WTPolI in pSBIK3 backbone</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of emrR, ugi, cda1 in pSBIK3 backbone (ASII)</li>
−
<li>Transformation in DH5&alpha;</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of EPPolI/ WTPolI in pSBIK3 backbone</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
 
<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#18">Week 18: (01.08.16-07.08.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#18">Week 18: (01.08.16-07.08.16): </button>  
 
<div id="18" class="collapse">  
 
<div id="18" class="collapse">  
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li>Colony PCR -> ASII did not work</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
−
<li>Repeated colony PCR -> sequencing of positive clones</li>
+
-> ASII did not work</li>
 +
<li>Repeated <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">colony PCR</a>
 +
-> sequencing of positive clones</li>
 
</ul>
 
</ul>
 
<br>
 
<br>
Line 287: Line 341:
 
<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#19">Week 19: (08.08.14-14.08.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#19">Week 19: (08.08.14-14.08.16): </button>  
 
<div id="19" class="collapse">  
 
<div id="19" class="collapse">  
 
<ul>
 
<ul>
−
<li>Transformation of pSBIC3:K584001 & K608010</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of pSBIC3:K584001 & K608010</li>
 
<li>Planning of reversion experiments</li>
 
<li>Planning of reversion experiments</li>
 
</ul>
 
</ul>
Line 296: Line 350:
 
<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#20">Week 20: (15.08.16-21.08.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#20">Week 20: (15.08.16-21.08.16): </button>  
 
<div id="20" class="collapse">  
 
<div id="20" class="collapse">  
 
<br>  
 
<br>  
 
<ul>
 
<ul>
 
<li>Sequencing results: mutagenesis ASI has mutations, ASII did not work</li>
 
<li>Sequencing results: mutagenesis ASI has mutations, ASII did not work</li>
−
<li>Transformation of ASII Gibson</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of ASII Gibson</li>
−
<li>Plasmid isolation of ASI/ ASII clones</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
−
<li>Colony PCR of ASII</li>
+
of ASI/ ASII clones</li>
−
<li>Restriction of positive clones of ASII</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
−
<li>Repeated colony PCR of ASI with cPCR_dnaQ_fw/VR -> plating out right clones of pSBIC3:dnaQ, pSBIC3:emrR</li>
+
of ASII</li>
−
<li>Restriction of right ASII clones with EcoR&thinsp;I/Pst&thinsp;I</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
−
<li>Restriction of right ASI clone with EcoR&thinsp;I/Pst&thinsp;I</li>
+
of positive clones of ASII</li>
 +
<li>Repeated <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">colony PCR</a>
 +
of ASI with primer cPCR_dnaQ_fw/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
-> plating out right clones of pSBIC3:dnaQ, pSBIC3:emrR</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of right ASII clones with EcoR&thinsp;I/Pst&thinsp;I</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of right ASI clone with EcoR&thinsp;I/Pst&thinsp;I</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
 
<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#21">Week 21: (22.08.16-28.08.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#21">Week 21: (22.08.16-28.08.16): </button>  
 
<div id="21" class="collapse">  
 
<div id="21" class="collapse">  
 
<br>  
 
<br>  
 
<ul>
 
<ul>
 
<li>Sequencing of dnaQ-dam-seqA (ASI), emrR_ugi_cda1 (ASII)</li>
 
<li>Sequencing of dnaQ-dam-seqA (ASI), emrR_ugi_cda1 (ASII)</li>
−
<li>PCR for coloning ASI into pSBIC3</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">PCR</a> for coloning ASI into pSBIC3</li>
 
<ul>
 
<ul>
 
<li>template pSBIK3:dnaq/dam/seqA, primer CH35/40, temp = 58.6&thinsp;&deg;C</li>
 
<li>template pSBIK3:dnaq/dam/seqA, primer CH35/40, temp = 58.6&thinsp;&deg;C</li>
−
<li>template pSBIC3:RFPcd, primer CH30/31, temp = 67&thinsp;&deg;C</li>
+
<li>template pSBIC3:RFP-cd, primer CH30/31, temp = 67&thinsp;&deg;C</li>
 
<li>template pSBIC3:AraC-Pbad, primer CH54/52, temp = 58.6&thinsp;&deg;C</li>
 
<li>template pSBIC3:AraC-Pbad, primer CH54/52, temp = 58.6&thinsp;&deg;C</li>
 
</ul>
 
</ul>
−
<li>DpnI digestion</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
−
<li>Restriction with EcoR&thinsp;I/Pst&thinsp;I</li>
+
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
with EcoR&thinsp;I/Pst&thinsp;I</li>
 
<ul>
 
<ul>
 
<li>pSBIK3:J04450</li>
 
<li>pSBIK3:J04450</li>
Line 330: Line 393:
 
<li> pSBIC3:K584001</li>
 
<li> pSBIC3:K584001</li>
 
</ul>
 
</ul>
−
<li>Plasmid isolation of positive emrR clones and positive ASII clone, strong RFP</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
−
<li>Restriction with EcoR&thinsp;I/Pst&thinsp;I of  ASII and pSBIC3:Strong RFP</li>
+
of positive emrR clones and positive ASII clone, strong RFP</li>
−
<li>Ligation of ASII and strong RFP</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
−
<li>Transformation of the ligation into DH5&alpha;</li>
+
with EcoR&thinsp;I/Pst&thinsp;I of  ASII and pSBIC3:Strong RFP</li>
−
<li>Gel extraction of pSBIK3, K608010, K584001</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> of ASII and strong RFP</li>
−
<li>Ligation of pSBIK3 + K608010</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">ligation</a> into DH5&alpha;</li>
−
<li>Overnight culture of the ligation</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
−
<li>Gel extraction of PCR products & PCR clean up</li>
+
of pSBIK3, K608010, K584001</li>
−
<li>Gibson assembly</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> of pSBIK3 + K608010</li>
 +
<li>Overnight culture of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">ligation</a></li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
of PCR products & PCR clean up</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a></li>
 
<ul>
 
<ul>
 
<li>pSBIC3:AraC-Pbad (non-leaky)</li>
 
<li>pSBIC3:AraC-Pbad (non-leaky)</li>
Line 344: Line 411:
 
<li>C3 backbone + ASI clone 15</li>
 
<li>C3 backbone + ASI clone 15</li>
 
</ul>
 
</ul>
−
<li>Ligation & Transformation in DH5&alpha;</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> & <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;</li>
−
<li>Colony PCR</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
</li>
 
<ul>
 
<ul>
−
<li>template pSBIC3:AraCPbad-nonleaky, primer VF/CH53, temp = 58&thinsp;&deg;C</li>
+
<li>template pSBIC3:AraCPbad-nonleaky, primer <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>/CH53, temp = 58&thinsp;&deg;C</li>
−
<li>template pSBIC3:ASI, primer VR/VF_rev, temp = 58&thinsp;&deg;C </li>
+
<li>template pSBIC3:ASI, primer <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/VF_rev, temp = 58&thinsp;&deg;C </li>
 
</ul>
 
</ul>
−
<li>Q5 PCR</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
</li>
 
<ul>
 
<ul>
 
<li> template A68T_A, primer CH20/A68T_rev, temp = 58.6&thinsp;&deg;C (AA)</li>
 
<li> template A68T_A, primer CH20/A68T_rev, temp = 58.6&thinsp;&deg;C (AA)</li>
Line 359: Line 429:
 
<li> template A380T_B, primer A380T_fw/CH21, temp = 58.1&thinsp;&deg;C (CB)</li>
 
<li> template A380T_B, primer A380T_fw/CH21, temp = 58.1&thinsp;&deg;C (CB)</li>
 
</ul>
 
</ul>
−
<li>Gel extraction</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
−
<li>Gibson assembly</li>
+
</li>
−
<li>Transformation in KRX</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a></li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in KRX</li>
 
<li>Sequencing of pSBIK3:EPPolI, pSBIK3:WTPolI with CH61,63,64</li>
 
<li>Sequencing of pSBIK3:EPPolI, pSBIK3:WTPolI with CH61,63,64</li>
−
<li>Colony PCR of AraC-Pbad (non leaky), ASI</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
−
<li>Q5 PCR template ASI, primer dnaQ_Gib_fw/ seqA_Gib_rev, temp = 57.5&thinsp;&deg;C</li>
+
of AraC-Pbad (non leaky), ASI</li>
−
<li>Sequencing of pSBIC3:emrR-1/-2 with VR/VF</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
−
<li>Colony PCR of pSBIK3:K608010_A/B/C with CH50/VR, temp = 50&thinsp;&deg;C</li>
+
template ASI, primer dnaQ_Gib_fw/ seqA_Gib_rev, temp = 57.5&thinsp;&deg;C</li>
−
<li>Plasmid isolation of pSBIC3:AraC-Pbad (non-leaky), pSBIK3:608010</li>
+
<li>Sequencing of pSBIC3:emrR-1/-2 with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
−
<li>Restriction of C3 backbone with EcoR&thinsp;I/Pst&thinsp;I</li>
+
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li>
−
<li>Transformation of pSBIC3:K516031 in KRX</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
−
<li>cPCR of pSBIC3:K608010_StopA/B/C with VR/VF</li>
+
of pSBIK3:K608010_A/B/C with CH50/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
−
<li>Sequencing A with VF, B with VR and C with VR</li>
+
, temp = 50&thinsp;&deg;C</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
 +
of pSBIC3:AraC-Pbad (non-leaky), pSBIK3:608010</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of C3 backbone with EcoR&thinsp;I/Pst&thinsp;I</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of pSBIC3:K516031 in KRX</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of pSBIC3:K608010_StopA/B/C with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li>
 +
<li>Sequencing A with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>, B with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
and C with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
 
<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#22">Week 22: (29.08.16-04.09.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#22">Week 22: (29.08.16-04.09.16): </button>  
 
<div id="22" class="collapse">  
 
<div id="22" class="collapse">  
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li>Plasmid isolation of Stop-GFPs -> Sequencing</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
−
<li>Restriction</li>
+
of Stop-GFPs -> Sequencing</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
</li>
 
<ul>
 
<ul>
 
<li>pSBIC3:AraCPbad with Spe&thinsp;I, Pst&thinsp;I</li>
 
<li>pSBIC3:AraCPbad with Spe&thinsp;I, Pst&thinsp;I</li>
Line 390: Line 474:
 
</ul>
 
</ul>
 
<li>Designing of new primer</li>
 
<li>Designing of new primer</li>
−
<li>Gel extraction of the Restriction</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
−
<li>Ligation </li>
+
of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">restriction digestion</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> </li>
 
<ul>
 
<ul>
−
<li>C3:AraC-Pbad + RFP Gen</li>
+
<li>C3:AraC-Pbad + RFP-Generator</li>
−
<li>C3:AraC-Pbad(non-leaky) + RFP Gen</li>
+
<li>C3:AraC-Pbad(non-leaky) + RFP-Generator</li>
 
<li>K3:Mut-I + Mut II</li>
 
<li>K3:Mut-I + Mut II</li>
 
<li>C3 + MutII</li>
 
<li>C3 + MutII</li>
 
</ul>
 
</ul>
−
<li>Repeat of Restriction of ASII: template K3:ASII, EcoR&thinsp;I-Hf/Pst&thinsp;I</li>
+
<li>Repeat of <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">restriction digestion</a>
−
<li>Gel extraction of the restriction</li>
+
of ASII: template K3:ASII, EcoR&thinsp;I-Hf/Pst&thinsp;I</li>
−
<li>Ligation</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
−
<li>Transformation of the Ligation, pHSG-EPPolI, pHSG-WTPolI in KRX</li>
+
of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">restriction digestion</a>
−
<li>cPCR with VR/VR of C3:ASII, C3:AraC-Pbad-RBS-RFP, C3:AraC-Pbad(non-leaky)-RBS-RFP, K3:ASI+ASII</li>
+
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a></li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">ligation</a>, pHSG-EPPolI, pHSG-WTPolI in KRX</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
with V<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>
 +
of C3:ASII, C3:AraC-Pbad-RBS-RFP, C3:AraC-Pbad(non-leaky)-RBS-RFP, K3:ASI+ASII</li>
 
<li>Plating out C3:AraC-Pbad-RBS-RFP with arabinose or glucose</li>
 
<li>Plating out C3:AraC-Pbad-RBS-RFP with arabinose or glucose</li>
−
<li>Q5 PCR, temp = 57&thinsp;&deg;C</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
, temp = 57&thinsp;&deg;C</li>
 
<ul>
 
<ul>
−
<li>template C3:AraC-Pbad(non)-RBS-RFP, primer CH60/CH61= </li>
+
<li>template C3:AraC-Pbad(non)-RBS-RFP, primer CH60/CH61</li>
 
<li> template C3:AraC-Pbad(non)-RBS-RFP, primer CH56/CH57</li>
 
<li> template C3:AraC-Pbad(non)-RBS-RFP, primer CH56/CH57</li>
 
<li> template C3:dnaQ, primer CH54/CH55</li>
 
<li> template C3:dnaQ, primer CH54/CH55</li>
Line 414: Line 507:
 
<li> template pHSG-EPPolI, primer CH42/C43</li>
 
<li> template pHSG-EPPolI, primer CH42/C43</li>
 
</ul>
 
</ul>
−
<li>DpnI digestion</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
−
<li>Gel extraction</li>
+
</li>
−
<li>Gibson assembly</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a></li>
 
<ul>
 
<ul>
 
<li>damseqA + C3</li>
 
<li>damseqA + C3</li>
Line 426: Line 521:
 
<li>EPPolI-Expression + PolI-BB</li>
 
<li>EPPolI-Expression + PolI-BB</li>
 
</ul>
 
</ul>
−
<li>Transformation</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a></li>
−
<li>cPCR with VR/VF, temp = 57&thinsp;&deg;C</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>, temp = 57&thinsp;&deg;C</li>
 
<ul>
 
<ul>
 
<li>C3:AraC-Pbad(tight)-B0031-WTPolI</li>
 
<li>C3:AraC-Pbad(tight)-B0031-WTPolI</li>
Line 439: Line 536:
 
<li>Sequencing</li>
 
<li>Sequencing</li>
 
<li>Cultur of KRX with C3:AraC-Pbad(tight)-B0031-E1010 with L-Arabinose -> repeat with Top10</li>
 
<li>Cultur of KRX with C3:AraC-Pbad(tight)-B0031-E1010 with L-Arabinose -> repeat with Top10</li>
−
<li>Transformation of C3:AraC-Pbad(tight)-B0031-E1010 in Top10</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of C3:AraC-Pbad(tight)-B0031-E1010 in Top10</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
 
<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#23">Week 23: (05.09.16-11.09.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#23">Week 23: (05.09.16-11.09.16): </button>  
 
<div id="23" class="collapse">  
 
<div id="23" class="collapse">  
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li>cPCR of ugi-cda1 -> Sequencing of right clones</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
−
<li>Q5 PCR, temp = 58&thinsp;&deg;C</li>
+
of ugi-cda1 -> Sequencing of right clones</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
, temp = 58&thinsp;&deg;C</li>
 
<ul>
 
<ul>
 
<li>template C3:damseqA, primer CH71/72</li>
 
<li>template C3:damseqA, primer CH71/72</li>
Line 456: Line 555:
 
<li>template K3:Stopp GFP A+B, primer CH64/21</li>
 
<li>template K3:Stopp GFP A+B, primer CH64/21</li>
 
</ul>
 
</ul>
−
<li>DpnI digestion of the Q5 PCR</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
−
<li>Gel extraction of Stop A, Stop B</li>
+
of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
−
<li>Gibson assembly stop A + stop B</li>
+
</li>
−
<li>Restriction with EcoR&thinsp;I/Pst&thinsp;I ASI+ASII, GFP_Astop_2, GFP_Astop_2</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
−
<li>Transformation of C3:PRha, C3:PRha-GFPGen in KRX</li>
+
of Stop A, Stop B</li>
−
<li>Transformation of PRha-GFPGen in top10</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> stop A + stop B</li>
−
<li>Transformation of StoppA+B in DH5&alpha;</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
with EcoR&thinsp;I/Pst&thinsp;I ASI+ASII, GFP_Astop_2, GFP_Astop_2</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of C3:PRha, C3:PRha-GFP-Generator in KRX</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of PRha-GFP-Generator in top10</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of StoppA+B in DH5&alpha;</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
 
<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#24">Week 24: (12.09.16-18.09.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#24">Week 24: (12.09.16-18.09.16): </button>  
 
<div id="24" class="collapse">  
 
<div id="24" class="collapse">  
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li>Q5 PCR, temp = 58&deg;C</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
, temp = 58&deg;C</li>
 
<ul>
 
<ul>
 
<li>template C3:dnaQ, primer 54/55</li>
 
<li>template C3:dnaQ, primer 54/55</li>
Line 478: Line 582:
 
<li> template C3:ASI+ASII, primer 54/76</li>
 
<li> template C3:ASI+ASII, primer 54/76</li>
 
<li> template C3:K516031, primer 56/57</li>
 
<li> template C3:K516031, primer 56/57</li>
−
<li> template C3: K516031, primer 60/61</li>
+
<li> template C3:K516031, primer 60/61</li>
−
<li> template C3: K516031, primer 60/61</li>
+
<li> template C3:K516031, primer 60/61</li>
−
<li> template C3: K516031, primer 75/57</li>
+
<li> template C3:K516031, primer 75/57</li>
 
</ul>
 
</ul>
−
<li>DpnI digestion</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
−
<li>Gel extraction</li>
+
</li>
−
<li>Gibson assembly of seqA</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
−
<li>Transformation in DH5&alpha; of C3:seqA, K3:StopA+B</li>
+
</li>
−
<li>Q5 PCR of C3:damseqA with CH71/72, temp = 58&thinsp;&deg;C</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of seqA</li>
−
<li>DpnI digestion</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha; of C3:seqA, K3:StopA+B</li>
−
<li>Gibson assembly of dnaQ_Gen + BB, WTPolI_Gen + BB, EPPol + BB, Mp6_Gen + BB</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
−
<li>Transformation of the Gibson assembly in DH5&alpha;</li>
+
of C3:damseqA with CH71/72, temp = 58&thinsp;&deg;C</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of dnaQ_Gen + BB, WTPolI_Gen + BB, EPPol + BB, Mp6_Gen + BB</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> in DH5&alpha;</li>
 
<li>Overnight cultures of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP </li>
 
<li>Overnight cultures of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP </li>
 
<li>Cultures (+ 50mM Arabinose) of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP </li>
 
<li>Cultures (+ 50mM Arabinose) of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP </li>
−
<li>cPCR of dnaQ-Gen, MP6-Gen with VR/VF and CH77/VR</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of dnaQ-Gen, MP6-Gen with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a> and CH77/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
</li>
 
<li>Overnight cultures of B0031-RFP-Ter, K808000, B0032-RFP-Ter</li>
 
<li>Overnight cultures of B0031-RFP-Ter, K808000, B0032-RFP-Ter</li>
−
<li>Q5 PCR of C3:damseqA, primer 71/72, gradient 55-59&thinsp;&deg;C/li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
of C3:damseqA, primer 71/72, gradient 55-59&thinsp;&deg;C/li>
 
<li>Sequencing of stop A+B</li>
 
<li>Sequencing of stop A+B</li>
−
<li>Restriction of K3:ASII, C3:Terminator with EcoR&thinsp;I, Pst&thinsp;I</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
−
<li>Gibson assembly of M6 in RFPGen -> Transformation</li>
+
of K3:ASII, C3:Terminator with EcoR&thinsp;I, Pst&thinsp;I</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of M6 in RFPGen -> <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a></li>
 
<li>Test cultivation of a 96-well plate</li>
 
<li>Test cultivation of a 96-well plate</li>
−
<li>cPCR of M6-Gen -> Plasmid isolation</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
−
<li>Restriction of dnaQ-Gen with Xba&thinsp;I/Pst&thinsp;I</li>
+
of M6-Gen -> <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of dnaQ-Gen with Xba&thinsp;I/Pst&thinsp;I</li>
 
<li>araE-PCR with temp 58&thinsp;&deg;C</li>
 
<li>araE-PCR with temp 58&thinsp;&deg;C</li>
−
<li>Gibson assembly of araE</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of araE</li>
−
<li>PCR of DNA, primer 81/82 and C3:med RFP 83/84</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">PCR</a> of DNA, primer 81/82 and C3:med RFP 83/84</li>
−
<li>Gel extraction of dnaQ-Gen</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
−
<li>Ligation with C3:AraC-Pbad/C3:PRha</li>
+
of dnaQ-Gen</li>
−
<li>DpnI digestion</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> with C3:AraC-Pbad/C3:PRha</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
 +
</li>
 
<li>PCR clean up</li>
 
<li>PCR clean up</li>
−
<li>Gibson of araE-Insert + araE-backbone</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of araE-Insert + araE-backbone</li>
−
<li>Transformation of the ligation in KRX</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">ligation</a> in KRX</li>
−
<li>Restriction of C3:M6-Gen with EcoR&thinsp;I/Xba&thinsp;I, weak/RFP Gen with Xba&thinsp;I/Pst&thinsp;I</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
−
<li>cPCR of C3:araE-Device</li>
+
of C3:M6-Gen with EcoR&thinsp;I/Xba&thinsp;I, weak/RFP Gen with Xba&thinsp;I/Pst&thinsp;I</li>
−
<li>Gibson C3 backbone + ugi_cda1, C3 backbone + ASII</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
−
<li>Transformation of the Gibson assembly in DH5&alpha;</li>
+
of C3:araE-Device</li>
−
<li>cPCR the transformation, Prha-B0031-dnaQ-Ter with VR/VF</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of C3 backbone + ugi_cda1, C3 backbone + ASII</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the Gibson assembly in DH5&alpha;</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">transformation</a>, Prha-B0031-dnaQ-Ter with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li>
 
</ul>
 
</ul>
 
</div>
 
</div>
 
<br>
 
<br>
  
−
<button data-toggle="collapse" data-target="#25">Week 25: (19.09.16-25.09.16): </button>  
+
<button data-toggle="collapse" class="btn-block"  data-target="#25">Week 25: (19.09.16-25.09.16): </button>  
 
<div id="25" class="collapse">  
 
<div id="25" class="collapse">  
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li>Restriction of M6-Generator with Xba&thinsp;I/Pst&thinsp;I, C3:K808000, C3:Prha with Spe&thinsp;I/Pst&thinsp;I</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of M6-Generator with Xba&thinsp;I/Pst&thinsp;I, C3:K808000, C3:Prha with Spe&thinsp;I/Pst&thinsp;I</li>
 
<li>Clean up</li>
 
<li>Clean up</li>
−
<li>Ligation with backbone</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> with backbone</li>
−
<li>Transformation of ligation, C3:AraCPbad(tight)-dnaQ in Top10, plA230 in JS200 with EPPolI/WTPolI</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">ligation</a>, C3:AraCPbad(tight)-dnaQ in Top10, plA230 in JS200 with EPPolI/WTPolI</li>
−
<li> Transformation of plA230 in JS200 with EPPolI/WTPolI; overnight culture with 10 ml Cm, Kan, 37&thinsp;&deg;C</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of plA230 in JS200 with EPPolI/WTPolI; overnight culture with 10 ml Cm, Kan, 37&thinsp;&deg;C</li>
−
<li>Transformation of C3:AraC(tight)dnaQ + plA230 in Top10</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of C3:AraC(tight)dnaQ + plA230 in Top10</li>
 
<li>Sequencing of C3:AraCPbad-dnaQ, C3:PRha-dnaQ</li>
 
<li>Sequencing of C3:AraCPbad-dnaQ, C3:PRha-dnaQ</li>
 
<li>Overnight culture of JS200 + pHSG-Pol + pLA230; Top 10 C3:AraCPbad-dnaQ</li>
 
<li>Overnight culture of JS200 + pHSG-Pol + pLA230; Top 10 C3:AraCPbad-dnaQ</li>
 
<li>Inoculation of Top 10 + C3:AraCPbad-dnaQ to OD 0.1; 1-2&thinsp;h growth, +25mM arabinose/25mM glycose</li>
 
<li>Inoculation of Top 10 + C3:AraCPbad-dnaQ to OD 0.1; 1-2&thinsp;h growth, +25mM arabinose/25mM glycose</li>
−
<li>Transformation of JS200 EP/WT with plA230</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of JS200 EP/WT with plA230</li>
−
<li>cPCR of AraC-Pbad-M6; Prha-M6</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
−
<li>Transformation of plA230 in JS00 EP/WT</li>
+
of AraC-Pbad-M6; Prha-M6</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of plA230 in JS00 EP/WT</li>
 
<li>Rifampicillin assay</li>
 
<li>Rifampicillin assay</li>
 
<li>beta-lactamase Reversions assay</li>
 
<li>beta-lactamase Reversions assay</li>
−
<li>Transformation of plA230 + C3:Pbad(med)-dnaQ in Top10</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of plA230 + C3:Pbad(med)-dnaQ in Top10</li>
 
<li>Overnight cultures</li>
 
<li>Overnight cultures</li>
 
</ul>
 
</ul>
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<br>
 
<br>
 
<ul>
 
<ul>
−
<li>Transformation of Pbad(med)dnaQ + plA230; Pbad-M6 + pLA230 in Top10</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of Pbad(med)dnaQ + plA230; Pbad-M6 + pLA230 in Top10</li>
−
<li>Transformation of JS200 + EP + plA230; JS200 + WT + pLA230 -> liquid culture</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of JS200 + EP + plA230; JS200 + WT + pLA230 -> liquid culture</li>
 
<li>Isolation of JS200 + PolI+plA230</li>
 
<li>Isolation of JS200 + PolI+plA230</li>
 
<li>Inoculation of Top10 + pLA230 + Pbad(med)-dnaQ; Top10 + plA230 + Pbad-M6 -> growth till OD 0.7336 (M6)/ 0.568 (dnaQ)</li>
 
<li>Inoculation of Top10 + pLA230 + Pbad(med)-dnaQ; Top10 + plA230 + Pbad-M6 -> growth till OD 0.7336 (M6)/ 0.568 (dnaQ)</li>
 
<li>Inoculation of with 1) 25mM arabinose; 2) + 25mM glucose 3) isolation</li>
 
<li>Inoculation of with 1) 25mM arabinose; 2) + 25mM glucose 3) isolation</li>
−
<li>Restriction of the MiSeq probes: WT, EP, plA230, pLA230+dnaQ; pLA230+M6</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of the MiSeq probes: WT, EP, pLA230, pLA230+dnaQ; pLA230+M6</li>
 
<li>Cloning of Pbad(med)WT/EP in pSB4C5; araE-dev + Pbad-GFP; B0032-RFP + Pbad; M6 + Pbad(med)</li>
 
<li>Cloning of Pbad(med)WT/EP in pSB4C5; araE-dev + Pbad-GFP; B0032-RFP + Pbad; M6 + Pbad(med)</li>
−
<li>Transformation of K3:K608010 –WT; - StopA; -StopB; - StopC in Top10</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of K3:K608010 –WT; - StopA; -StopB; - StopC in Top10</li>
−
<li>Transformation of C3:Pbad(med)dnaQ & K3:K608010 –WT; - StopA; -StopB; - StopC in Top10</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of C3:Pbad(med)dnaQ & K3:K608010 –WT; - StopA; -StopB; - StopC in Top10</li>
 
<li>Reversions assay</li>
 
<li>Reversions assay</li>
−
<li>Restriction of E/P of pSB4C5(E/P)</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
−
<li>Q5 PCR</li>
+
of E/P of pSB4C5(E/P)</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
</li>
 
<ul>
 
<ul>
−
<li>template C3:ugi-cda, primer 67/68, temp = 58&thinsp;&deg;C</li>
+
<li>template C3:ugi-cda, primer <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
, temp = 58&thinsp;&deg;C</li>
 
<li>template C3:cda, primer 69/70, temp = 60&thinsp;&deg;C</li>
 
<li>template C3:cda, primer 69/70, temp = 60&thinsp;&deg;C</li>
 
<li>template A3, primer CK06/05, temp = 59.9&thinsp;&deg;C</li>
 
<li>template A3, primer CK06/05, temp = 59.9&thinsp;&deg;C</li>
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<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li>Transformation of C3:dnaQ Dev + pLA230, C3:M6 Dev + pLA230 in Top10</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of C3:dnaQ Dev + pLA230, C3:M6 Dev + pLA230 in Top10</li>
 
<li>Reversions assay</li>
 
<li>Reversions assay</li>
−
<li></li>
 
−
<li></li>
 
−
<li></li>
 
−
<li></li>
 
−
 
 
</ul>
 
</ul>
 
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<br>
 
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Latest revision as of 16:11, 1 December 2016



Lab Notebook

Documenting the Mutation

Mutation



Firstly, we researched for literature on issues involving in vivo mutagenesis and error prone polymerase. In this phase we read the papers from Prof. Dr. Manel Camps.


Discussing the results of the literature research and creating a first draft of what must be done in the laboratory. Further literature research.


Looking for suitable parts that can be used for our mutagenesis system. Creating plasmid circuits in geneious. Extension of the first draft.


Designing of first primers and getting to know the laboratory. Also literature research.


Contacting the leading expert for the error prone polymerase via e-mail. Dividing the laboratory tasks and creating milestones.


  • Transformation in DH5α:
    • pHSG-WTPolI
    • pHSG-EPPolI
    • pLA230
    • pSBIK3:J04450
    • pSBIA3:E0044
  • Everything besides pSBIA3:E0044 grew. Taking overnight cultures at 37 °C.
  • Plating provided JS200 strain out.


  • Plasmid isolation of:
    • pHSG-WTPolI
    • pHSG-EPPolI
    • pLA230
    • pSBIK3:J04450
  • Overnight cultures from JS200.
  • JS200 glycerin stocks (500 μl culture + 200 μl 86% glycerin)


Rethinking of our plan.


  • Transformation in DH5α:
    • pSBIC3:E1010
    • pSBIC3:K731722
  • Transformation in JS200;:
    • pHSG-EPPolI & pLA230
    • pHSG-WTPolI & pLA230
  • Platting out pSBIC3:Isopropanolpathway from iGEM Bielefeld 2014


  • Overnight cultures of transformation from week 9.
  • Plasmid isolation
  • Plasmid isolation & restriction digestion with Spe I.
  • Gel
  • Idea: BioBrick site directly behind pMBi ori of pSBIK3
    • 1. Deletion of BioBrick site
      • Q5 PCR
        • Template: pSBIK3:J04450
        • Part 1 with primer pSB1K3_Del_for /pSB1K3_D_for_P2 , temp = 56 °C
        • Part 2 with primer pSB1K3_Del_rev /pSB1K3_D_rev_P1 , temp = 57 °C
        • -> Part 1 worked; part 2 not
      • Transformation in DH5α of pSBIC3:K592100
    • 2.: New BioBrick site behind ori




  • Q5 PCR at 67 °C:
    • template: pSBIK3:J04450; primer PS02/PS03
    • template: pSBIC3:E1010; primer CH30/CH31
    • template: pSBIC3:K592100; primer PS00/PS01
  • Gibson assembly: 15 μl Gibson Mastermix + 3.5 μl pSBIK3 Backbone + 1.5 μl BFP-Insert
  • Transformation in DH5α
  • 12 clones on LB-Kan plate that glow blue!


  • Gene synthesis is there (AraProm, dnaQ, dam-seqA, emrR, ugi-cda1)! Resolve in TE-buffer
  • Gibson assembly of the gene synthesis in pSBIC3 backbone
  • Transformation in NEB electro competent cells
  • Colony PCR with VR/VF
  • Overnight culture from AraProm, dam-seqA, emrR


  • Plasmid isolation of the overnight cultures
  • Restriction digestion with EcoR I/ Pst I
  • Sequencing of AraProm, dam-seqA, emrR
  • Transformation in DH5α of pSBIC3:K516132
  • Repeated Colony PCR of dnaQ, ugi-cda1 -> only empty plasmids -> new primer
  • overnight culture of pSBIC3:K516132
  • Plasmid isolation of pSBIC3:K516132
  • Q5 PCR with template pSBIC3:K516132, primer CH30/CH31, temp = 67 °C
  • DPN1 digestion of pcr product pSBIC3:K516132, 1h 37 °C
  • 1% Agarosegel & purification of the 2 kb band -> new backbone
  • repeated Gibson assembly with the new backbone and ugi-cda1/ dnaQ
  • Transformation in DH5α
    • pSBIA3:J04450
    • pSBIC3:J06702
    • GFP-cd
    • pSBIC:E2050
    • dnaQ Gibson
    • ugi-cda1 Gibson
  • Overnight culture of DH5α with pSBIK3:RFP-cd/ pSBIK3:BFP-cd
  • Testing of the fluorescent proteins RFP and BFP with the Teacan
  • Colony PCR of pSBIC3:dnaQ with VR/VF
  • Overnight culture of good clones, pGFPuv, pSBIA3:J04450, pSBIC3:J06702


  • Primer design for genesynthesis
  • Plasmid isolation of the overnight cultures
  • Restriction digestion of pSBIC3:dnaQ, mCherry, E2050-cds
  • Sequencing of dnaQ clones with VR /VF
  • Colony PCR with CH34/VR on ugi-cda1
  • Sequencing of ugi-cda1
  • Assembly of dnaQ, dam, seqA
    • Q5 PCR
      • template pSBIC3:dnaQ, primer dnaQ_Gib_for /dnaQ_Gib_rev , temp = 59.9 °C
      • template pSBIC3:dam-seqA, primer dam_Gib_for /dam_Gib_rev , temp = 58.3 °C
      • template pSBIC3:dam-seqA, primer CH39/CH40, temp = 57 °
      • template pHSG-WTPolI, primer PolI_Gib_for /PolI_Gib_rev , temp = 58.4 °C
      • template pHSG-EPPolI, primer PolI_Gib_for /PolI_Gib_rev , temp = 58.4 °C
    • DPN1 digestion , Gel extraction
    • Restriction digestion of pSBIC3:RFP-cd with Xba I/Spe I, gel extraction of 2 kb band




  • Colony PCR of ASI with VR /VF -> plating positive clones out
  • Sequencing of positive clones
  • Q5 PCR
    • template pSBIC3:emrR, primer BF1/2
    • template pSBIC3:ugi-cda1, primer BF4/5
    • template pSBIC3:emrR, primer BF6/7
    • template pHSG:EPPolI, primer CH42/43
    • template pHSG:WTPolI, primer CH42/43
  • PCR clean up of the Q5 PCR
  • Gibson assembly of emrR, ugi, cda1 in pSBIK3 backbone (ASII)
  • Gibson assembly of EPPolI/ WTPolI in pSBIK3 backbone
  • Transformation in DH5α




  • Transformation of pSBIC3:K584001 & K608010
  • Planning of reversion experiments




  • Sequencing of dnaQ-dam-seqA (ASI), emrR_ugi_cda1 (ASII)
  • PCR for coloning ASI into pSBIC3
    • template pSBIK3:dnaq/dam/seqA, primer CH35/40, temp = 58.6 °C
    • template pSBIC3:RFP-cd, primer CH30/31, temp = 67 °C
    • template pSBIC3:AraC-Pbad, primer CH54/52, temp = 58.6 °C
  • DPN1 digestion
  • Restriction digestion with EcoR I/Pst I
    • pSBIK3:J04450
    • pSBIC3:K608010
    • pSBIC3:K584001
  • Plasmid isolation of positive emrR clones and positive ASII clone, strong RFP
  • Restriction digestion with EcoR I/Pst I of ASII and pSBIC3:Strong RFP
  • Ligation of ASII and strong RFP
  • Transformation of the ligation into DH5α
  • Gel extraction of pSBIK3, K608010, K584001
  • Ligation of pSBIK3 + K608010
  • Overnight culture of the ligation
  • Gel extraction of PCR products & PCR clean up
  • Gibson assembly
    • pSBIC3:AraC-Pbad (non-leaky)
    • C3 backbone + ASI clone 4
    • C3 backbone + ASI clone 15
  • Ligation & Transformation in DH5α
  • Colony PCR
    • template pSBIC3:AraCPbad-nonleaky, primer VF/CH53, temp = 58 °C
    • template pSBIC3:ASI, primer VR /VF_rev, temp = 58 °C
  • Q5 PCR
    • template A68T_A, primer CH20/A68T_rev, temp = 58.6 °C (AA)
    • template A68T_B, primer A68T_B/CH21, temp = 58.5 °C (AB)
    • template C379A_A, primer CH20/C379A_rev, temp = 57.7 °C (BA)
    • template C379A_B, primer C379A_fw/CH21, temp = 58 °C (BB)
    • template A380T_A, primer CH20/ A380T_rev, temp = 58.1 °C (CA)
    • template A380T_B, primer A380T_fw/CH21, temp = 58.1 °C (CB)
  • Gel extraction
  • Gibson assembly
  • Transformation in KRX
  • Sequencing of pSBIK3:EPPolI, pSBIK3:WTPolI with CH61,63,64
  • Colony PCR of AraC-Pbad (non leaky), ASI
  • Q5 PCR template ASI, primer dnaQ_Gib_fw/ seqA_Gib_rev, temp = 57.5 °C
  • Sequencing of pSBIC3:emrR-1/-2 with VR /VF
  • Colony PCR of pSBIK3:K608010_A/B/C with CH50/VR , temp = 50 °C
  • Plasmid isolation of pSBIC3:AraC-Pbad (non-leaky), pSBIK3:608010
  • Restriction digestion of C3 backbone with EcoR I/Pst I
  • Transformation of pSBIC3:K516031 in KRX
  • Colony PCR of pSBIC3:K608010_StopA/B/C with VR /VF
  • Sequencing A with VF, B with VR and C with VR


  • Plasmid isolation of Stop-GFPs -> Sequencing
  • Restriction digestion
    • pSBIC3:AraCPbad with Spe I, Pst I
    • pSBIC3:AraCPbad(non-leaky) with Spe I, Pst I
    • pSBIC3:RFP Generator with Xba I, Pst I
    • pSBIK3:MP6-I-Klon 4 with Spe I, Pst I
    • pSBIK3:MP6-II-Klon with Xba I, Pst I
  • Designing of new primer
  • Gel extraction of the restriction digestion
  • Ligation
    • C3:AraC-Pbad + RFP-Generator
    • C3:AraC-Pbad(non-leaky) + RFP-Generator
    • K3:Mut-I + Mut II
    • C3 + MutII
  • Repeat of restriction digestion of ASII: template K3:ASII, EcoR I-Hf/Pst I
  • Gel extraction of the restriction digestion
  • Ligation
  • Transformation of the ligation, pHSG-EPPolI, pHSG-WTPolI in KRX
  • Colony PCR with VVR /VF of C3:ASII, C3:AraC-Pbad-RBS-RFP, C3:AraC-Pbad(non-leaky)-RBS-RFP, K3:ASI+ASII
  • Plating out C3:AraC-Pbad-RBS-RFP with arabinose or glucose
  • Q5 PCR , temp = 57 °C
    • template C3:AraC-Pbad(non)-RBS-RFP, primer CH60/CH61
    • template C3:AraC-Pbad(non)-RBS-RFP, primer CH56/CH57
    • template C3:dnaQ, primer CH54/CH55
    • template pHSG-WTPolI, primer CH58/C59
    • template pHSG-WTPolI, primer CH42/C43
    • template pHSG-EPPolI, primer CH58/C59
    • template pHSG-EPPolI, primer CH42/C43
  • DPN1 digestion
  • Gel extraction
  • Gibson assembly
    • damseqA + C3
    • ugi-cda1 + C3
    • WTPolI-Part + C3
    • EPPolI-Part + C3
    • dnaQ-Insert + dnaQ-BB
    • WTPolI-Expression + PolI-BB
    • EPPolI-Expression + PolI-BB
  • Transformation
  • Colony PCR with VR /VF, temp = 57 °C
    • C3:AraC-Pbad(tight)-B0031-WTPolI
    • C3:ugi-cda1
    • C3:AraC-Pbad(tight)-B0031-dnaQ
    • C3:EPPolI
    • C3:AraC-Pbad(tight)-B0031-EPPolI
    • C3:seqA
    • C3:WTPolI
  • Sequencing
  • Cultur of KRX with C3:AraC-Pbad(tight)-B0031-E1010 with L-Arabinose -> repeat with Top10
  • Transformation of C3:AraC-Pbad(tight)-B0031-E1010 in Top10






  • Restriction digestion of M6-Generator with Xba I/Pst I, C3:K808000, C3:Prha with Spe I/Pst I
  • Clean up
  • Ligation with backbone
  • Transformation of ligation, C3:AraCPbad(tight)-dnaQ in Top10, plA230 in JS200 with EPPolI/WTPolI
  • Transformation of plA230 in JS200 with EPPolI/WTPolI; overnight culture with 10 ml Cm, Kan, 37 °C
  • Transformation of C3:AraC(tight)dnaQ + plA230 in Top10
  • Sequencing of C3:AraCPbad-dnaQ, C3:PRha-dnaQ
  • Overnight culture of JS200 + pHSG-Pol + pLA230; Top 10 C3:AraCPbad-dnaQ
  • Inoculation of Top 10 + C3:AraCPbad-dnaQ to OD 0.1; 1-2 h growth, +25mM arabinose/25mM glycose
  • Transformation of JS200 EP/WT with plA230
  • Colony PCR of AraC-Pbad-M6; Prha-M6
  • Transformation of plA230 in JS00 EP/WT
  • Rifampicillin assay
  • beta-lactamase Reversions assay
  • Transformation of plA230 + C3:Pbad(med)-dnaQ in Top10
  • Overnight cultures



  • Transformation of Pbad(med)dnaQ + plA230; Pbad-M6 + pLA230 in Top10
  • Transformation of JS200 + EP + plA230; JS200 + WT + pLA230 -> liquid culture
  • Isolation of JS200 + PolI+plA230
  • Inoculation of Top10 + pLA230 + Pbad(med)-dnaQ; Top10 + plA230 + Pbad-M6 -> growth till OD 0.7336 (M6)/ 0.568 (dnaQ)
  • Inoculation of with 1) 25mM arabinose; 2) + 25mM glucose 3) isolation
  • Restriction digestion of the MiSeq probes: WT, EP, pLA230, pLA230+dnaQ; pLA230+M6
  • Cloning of Pbad(med)WT/EP in pSB4C5; araE-dev + Pbad-GFP; B0032-RFP + Pbad; M6 + Pbad(med)
  • Transformation of K3:K608010 –WT; - StopA; -StopB; - StopC in Top10
  • Transformation of C3:Pbad(med)dnaQ & K3:K608010 –WT; - StopA; -StopB; - StopC in Top10
  • Reversions assay
  • Restriction digestion of E/P of pSB4C5(E/P)
  • Q5 PCR
    • template C3:ugi-cda, primer VF/VR , temp = 58 °C
    • template C3:cda, primer 69/70, temp = 60 °C
    • template A3, primer CK06/05, temp = 59.9 °C
    • template reporter, primer CK07/08, temp = 59 °C



  • Transformation of C3:dnaQ Dev + pLA230, C3:M6 Dev + pLA230 in Top10
  • Reversions assay


  • Analysis reversion assays
  • Analysis of MiSeq data