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

Line 65: Line 65:
 
<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>
Line 110: Line 111:
 
<ul>
 
<ul>
 
<li> Overnight cultures of <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">transformation</a>  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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid</a> Isolation</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
−
Plasmid isolation & <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">restriction digestion</a></li>
+
</li>
−
  with Spe&thinsp;I. </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>
Line 118: Line 121:
 
<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>
Line 138: Line 142:
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li><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">Restriction digestion</a>
 
  of pSBIK3:J04450 with Spe&thinsp;I and Xba&thinsp;I</li>
 
  of pSBIK3:J04450 with Spe&thinsp;I and Xba&thinsp;I</li>
−
<li>Colony PCR of K592100</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>
Line 150: Line 155:
 
<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 170: Line 176:
 
<li>Gibson assembly of the gene synthesis in pSBIC3 backbone</li>
 
<li>Gibson assembly of the gene synthesis in pSBIC3 backbone</li>
 
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> 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 VR/VF</li>
 
<li>Overnight culture from AraProm, dam-seqA, emrR</li>
 
<li>Overnight culture from AraProm, dam-seqA, emrR</li>
 
</ul>
 
</ul>
Line 180: Line 187:
 
<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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a></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>
 
  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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> 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 Gibson assembly with the new backbone and ugi-cda1/ dnaQ</li>
Line 203: Line 215:
 
<li>Overnight culture of DH5&alpha; with pSBIK3:RFP-cd/ pSBIK3:BFP-cd </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 VR/VF</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>
Line 214: Line 227:
 
<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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a></li>
+
of the overnight cultures</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 
  of pSBIC3:dnaQ, mCherry, E2050-cds</li>
 
  of pSBIC3:dnaQ, mCherry, E2050-cds</li>
 
<li>Sequencing of dnaQ clones with VR/VF</li>
 
<li>Sequencing of dnaQ clones with VR/VF</li>
−
<li>Colony-PCR with CH34/VR on ugi-cda1</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
with CH34/VR 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 CH35/CH36, temp = 59.9&thinsp;&deg;C</li>
Line 230: Line 246:
 
<li>template pHSG-EPPolI, primer CH42/CH43, temp = 58.4&thinsp;&deg;C</li>
 
<li>template pHSG-EPPolI, primer CH42/CH43, 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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a></li>
+
, <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
−
  of pSBIC3:RFP-cd with Xba&thinsp;I/Spe&thinsp;I, gel extraction of 2&thinsp;kb band</li>
+
<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 244: Line 262:
 
<li>Gibson of dnaQ, dam, seqA, C3 backbone</li>
 
<li>Gibson of dnaQ, dam, seqA, C3 backbone</li>
 
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> 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 252: Line 272:
 
<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>
 +
and <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">gel extraction</a>
 +
of pSBIK3 backbone</li>
 
<li>Gibson assembly of dnaQ, dam, seqA in K3 backbone (ASI)</li>
 
<li>Gibson assembly 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>
 
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;</li>
Line 263: Line 285:
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li>Colony-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 VR/VF -> 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 273: Line 297:
 
<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>
 
<li>Gibson assembly of emrR, ugi, cda1 in pSBIK3 backbone (ASII)</li>
 
<li>Gibson assembly of emrR, ugi, cda1 in pSBIK3 backbone (ASII)</li>
 
<li>Gibson assembly of EPPolI/ WTPolI in pSBIK3 backbone</li>
 
<li>Gibson assembly of EPPolI/ WTPolI in pSBIK3 backbone</li>
Line 285: Line 310:
 
<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 307: Line 334:
 
<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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> 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><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">Colony PCR</a>
 +
of ASII</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 
  of positive clones of ASII</li>
 
  of positive clones of ASII</li>
−
<li>Repeated colony PCR of ASI with primer cPCR_dnaQ_fw/VR -> plating out right clones of pSBIC3:dnaQ, pSBIC3:emrR</li>
+
<li>Repeated <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">colony PCR</a>
−
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a></li>
+
of ASI with primer cPCR_dnaQ_fw/VR -> 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>
 
  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>
+
<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>
 
  of right ASI clone with EcoR&thinsp;I/Pst&thinsp;I</li>
 
</ul>
 
</ul>
Line 331: Line 361:
 
<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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a></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>
 
  with EcoR&thinsp;I/Pst&thinsp;I</li>
 
<ul>
 
<ul>
Line 339: Line 370:
 
<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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a></li>
+
of positive emrR clones and positive ASII clone, strong RFP</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 
  with EcoR&thinsp;I/Pst&thinsp;I of  ASII and pSBIC3:Strong RFP</li>
 
  with EcoR&thinsp;I/Pst&thinsp;I of  ASII and pSBIC3:Strong RFP</li>
−
<li>Ligation of ASII and strong RFP</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> of ASII and strong RFP</li>
−
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the ligation into DH5&alpha;</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>Gel extraction of pSBIK3, K608010, K584001</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
−
<li>Ligation of pSBIK3 + K608010</li>
+
of pSBIK3, K608010, K584001</li>
−
<li>Overnight culture of the ligation</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> of pSBIK3 + K608010</li>
−
<li>Gel extraction of PCR products & PCR clean up</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>Gibson assembly</li>
 
<li>Gibson assembly</li>
 
<ul>
 
<ul>
Line 354: Line 388:
 
<li>C3 backbone + ASI clone 15</li>
 
<li>C3 backbone + ASI clone 15</li>
 
</ul>
 
</ul>
−
<li>Ligation & <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> 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 VF/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 VR/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 369: Line 405:
 
<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>
 
<li>Gibson assembly</li>
 
<li>Gibson assembly</li>
 
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in KRX</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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
template ASI, primer dnaQ_Gib_fw/ seqA_Gib_rev, temp = 57.5&thinsp;&deg;C</li>
 
<li>Sequencing of pSBIC3:emrR-1/-2 with VR/VF</li>
 
<li>Sequencing of pSBIC3:emrR-1/-2 with VR/VF</li>
−
<li>Colony PCR of pSBIK3:K608010_A/B/C with CH50/VR, temp = 50&thinsp;&deg;C</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
−
<li>Plasmid isolation of pSBIC3:AraC-Pbad (non-leaky), pSBIK3:608010</li>
+
of pSBIK3:K608010_A/B/C with CH50/VR, temp = 50&thinsp;&deg;C</li>
−
<li><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">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>
 
  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">Transformation</a> of pSBIC3:K516031 in KRX</li>
−
<li>cPCR of pSBIC3:K608010_StopA/B/C with VR/VF</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of pSBIC3:K608010_StopA/B/C with VR/VF</li>
 
<li>Sequencing A with VF, B with VR and C with VR</li>
 
<li>Sequencing A with VF, B with VR and C with VR</li>
 
</ul>
 
</ul>
Line 391: Line 433:
 
<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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a></li>
+
of Stop-GFPs -> Sequencing</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 
</li>
 
</li>
 
<ul>
 
<ul>
Line 402: Line 445:
 
</ul>
 
</ul>
 
<li>Designing of new primer</li>
 
<li>Designing of new primer</li>
−
<li>Gel extraction 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">Gel extraction</a>
 +
of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">restriction digestion</a>
 
</li>
 
</li>
−
<li>Ligation </li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> </li>
 
<ul>
 
<ul>
 
<li>C3:AraC-Pbad + RFP-Generator</li>
 
<li>C3:AraC-Pbad + RFP-Generator</li>
Line 411: Line 455:
 
<li>C3 + MutII</li>
 
<li>C3 + MutII</li>
 
</ul>
 
</ul>
−
<li>Repeat of <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">restriction digestion</a></li>
+
<li>Repeat of <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">restriction digestion</a>
 
  of ASII: template K3:ASII, EcoR&thinsp;I-Hf/Pst&thinsp;I</li>
 
  of ASII: template K3:ASII, EcoR&thinsp;I-Hf/Pst&thinsp;I</li>
−
<li>Gel extraction 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">Gel extraction</a>
 +
of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">restriction digestion</a>
 
</li>
 
</li>
−
<li>Ligation</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 Ligation, pHSG-EPPolI, pHSG-WTPolI 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>, pHSG-EPPolI, pHSG-WTPolI in KRX</li>
−
<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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
with VR/VR 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>
Line 429: Line 476:
 
<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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
</li>
 
<li>Gibson assembly</li>
 
<li>Gibson assembly</li>
 
<ul>
 
<ul>
Line 442: Line 491:
 
</ul>
 
</ul>
 
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a></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 VR/VF, 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 463: Line 513:
 
<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 471: Line 523:
 
<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>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
of Stop A, Stop B</li>
 
<li>Gibson assembly stop A + stop B</li>
 
<li>Gibson assembly stop A + stop B</li>
−
<li><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">Restriction digestion</a>
 
  with EcoR&thinsp;I/Pst&thinsp;I ASI+ASII, GFP_Astop_2, GFP_Astop_2</li>
 
  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 C3:PRha, C3:PRha-GFP-Generator in KRX</li>
Line 487: Line 542:
 
<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 498: Line 554:
 
<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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
</li>
 
<li>Gibson assembly of seqA</li>
 
<li>Gibson assembly of seqA</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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha; of C3:seqA, K3:StopA+B</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">Q5 PCR</a>
−
<li>DpnI digestion</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>Gibson assembly of dnaQ_Gen + BB, WTPolI_Gen + BB, EPPol + BB, Mp6_Gen + BB</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">Transformation</a> of the Gibson assembly in DH5&alpha;</li>
 
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the Gibson assembly 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 VR/VF and CH77/VR</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><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">Restriction digestion</a>
 
  of K3:ASII, C3:Terminator with EcoR&thinsp;I, Pst&thinsp;I</li>
 
  of K3:ASII, C3:Terminator with EcoR&thinsp;I, Pst&thinsp;I</li>
 
<li>Gibson assembly of M6 in RFPGen -> <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a></li>
 
<li>Gibson assembly 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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a></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>
 
  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>Gibson assembly of araE</li>
 
<li>PCR of DNA, primer 81/82 and C3:med RFP 83/84</li>
 
<li>PCR 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>Gibson of araE-Insert + araE-backbone</li>
−
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> 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><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">Restriction digestion</a>
 
  of C3:M6-Gen with EcoR&thinsp;I/Xba&thinsp;I, weak/RFP Gen with Xba&thinsp;I/Pst&thinsp;I</li>
 
  of C3:M6-Gen with EcoR&thinsp;I/Xba&thinsp;I, weak/RFP Gen with Xba&thinsp;I/Pst&thinsp;I</li>
−
<li>cPCR of C3:araE-Device</li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of C3:araE-Device</li>
 
<li>Gibson C3 backbone + ugi_cda1, C3 backbone + ASII</li>
 
<li>Gibson 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">Transformation</a> of the Gibson assembly in DH5&alpha;</li>
−
<li>cPCR the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">transformation</a>, Prha-B0031-dnaQ-Ter with VR/VF</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 VR/VF</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
Line 542: Line 610:
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li><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">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>
 
  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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> 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><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><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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> 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>
Line 553: Line 621:
 
<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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> 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>
 +
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><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>
Line 573: Line 642:
 
<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><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">Restriction digestion</a>
 
  of the MiSeq probes: WT, EP, pLA230, pLA230+dnaQ; pLA230+M6</li>
 
  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>
Line 579: Line 648:
 
<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><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><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">Restriction digestion</a>
 
  of E/P of pSB4C5(E/P)</li>
 
  of E/P of pSB4C5(E/P)</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 C3:ugi-cda, primer 67/68, temp = 58&thinsp;&deg;C</li>
 
<li>template C3:ugi-cda, primer 67/68, temp = 58&thinsp;&deg;C</li>

Revision as of 17:50, 19 October 2016



lab notebook.

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






  • 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 CH35/CH36, temp = 59.9 °C
      • template pSBIC3:dam-seqA, primer CH37/CH38, temp = 58.3 °C
      • template pSBIC3:dam-seqA, primer CH39/CH40, temp = 57 °
      • template pHSG-WTPolI, primer CH42/CH43, temp = 58.4 °C
      • template pHSG-EPPolI, primer CH42/CH43, 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


  • Gibson of dnaQ, dam, seqA, C3 backbone
  • Transformation in KRX compis
  • Colony PCR with the Gibson clones -> no bands
  • Q5 PCR
    • template pSBIC3:dnaQ, primer CH35/36, temp = 59.9 °C
    • template pSBIC3:dam-seqA, primer CH37/38, temp = 58.3 °C
    • template pSBIC3:dam-seqA, primer CH39/40, temp = 57 °C
    • template pSBIK3:J04450, primer CH30/31, temp = 67 °C
  • DPN1 digestion and gel extraction of pSBIK3 backbone
  • Gibson assembly of dnaQ, dam, seqA in K3 backbone (ASI)
  • Transformation in DH5α


  • 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 VR/VR 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




  • Q5 PCR , temp = 58°C
    • template C3:dnaQ, primer 54/55
    • template C3:WTPolI (CWP1), primer 58/59
    • template C3:EPPolI (CEP1), primer 58/59
    • template C3:ASI+ASII, primer 54/76
    • template C3:K516031, primer 56/57
    • template C3:K516031, primer 60/61
    • template C3:K516031, primer 60/61
    • template C3:K516031, primer 75/57
  • DPN1 digestion
  • Gel extraction
  • Gibson assembly of seqA
  • Transformation in DH5α of C3:seqA, K3:StopA+B
  • Q5 PCR of C3:damseqA with CH71/72, temp = 58 °C
  • DPN1 digestion
  • Gibson assembly of dnaQ_Gen + BB, WTPolI_Gen + BB, EPPol + BB, Mp6_Gen + BB
  • Transformation of the Gibson assembly in DH5α
  • Overnight cultures of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP
  • Cultures (+ 50mM Arabinose) of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP
  • Colony PCR of dnaQ-Gen, MP6-Gen with VR/VF and CH77/VR
  • Overnight cultures of B0031-RFP-Ter, K808000, B0032-RFP-Ter
  • Q5 PCR of C3:damseqA, primer 71/72, gradient 55-59 °C/li>
  • Sequencing of stop A+B
  • Restriction digestion of K3:ASII, C3:Terminator with EcoR I, Pst I
  • Gibson assembly of M6 in RFPGen -> Transformation
  • Test cultivation of a 96-well plate
  • Colony PCR of M6-Gen -> Plasmid isolation
  • Restriction digestion of dnaQ-Gen with Xba I/Pst I
  • araE-PCR with temp 58 °C
  • Gibson assembly of araE
  • PCR of DNA, primer 81/82 and C3:med RFP 83/84
  • Gel extraction of dnaQ-Gen
  • Ligation with C3:AraC-Pbad/C3:PRha
  • DPN1 digestion
  • PCR clean up
  • Gibson of araE-Insert + araE-backbone
  • Transformation of the ligation in KRX
  • Restriction digestion of C3:M6-Gen with EcoR I/Xba I, weak/RFP Gen with Xba I/Pst I
  • Colony PCR of C3:araE-Device
  • Gibson C3 backbone + ugi_cda1, C3 backbone + ASII
  • Transformation of the Gibson assembly in DH5α
  • Colony PCR the transformation, Prha-B0031-dnaQ-Ter with VR/VF


  • 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 67/68, 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