Difference between revisions of "Team:Tuebingen/Notebook"

Line 78: Line 78:
 
<p>  
 
<p>  
 
Ligation of gene blocks (Cre, Cre_sn, Dronpa, HCV, HCV+linker, nluc), linker L6, L9, L12, L15, nDegron and NLS with pSB1C3 and transformation in DH10beta: </p>
 
Ligation of gene blocks (Cre, Cre_sn, Dronpa, HCV, HCV+linker, nluc), linker L6, L9, L12, L15, nDegron and NLS with pSB1C3 and transformation in DH10beta: </p>
 +
<p>Colony PCR and test restriction to check for correct transformation</p>
 
<p>
 
<p>
 
pSB-Cre_sn and pSB-Luciferase did not yield correct colonies (transformation repeated), the other constructs resulted in correct colonies
 
pSB-Cre_sn and pSB-Luciferase did not yield correct colonies (transformation repeated), the other constructs resulted in correct colonies
Line 90: Line 91:
 
<div id="dia1" class="dia cal">
 
<div id="dia1" class="dia cal">
 
<h2>Cre-Dronpa</h2>
 
<h2>Cre-Dronpa</h2>
−
<h2>Cre Reporter Cassette</h2>
+
<p>Colony PCR of pSB-luc</p>
−
<h2>pRS Vectors</h2>
+
<p>Transformation of pSB-Gal and pSB-luc in DH10beta</p>
−
<h2>Sensors/Promotors</h2>
+
<p>Three part assembly of Linker(6/9/12/15) + Cre, Linker(6,9,12,15) + Dronpa with pTUM100 and transformation in yeast resulting in mixed colony PCR results (L6-Cre, L9-Cre, L12-Cre and L9-Dronpa yielded correct bands, the other assamblies did not result in correct fragment lengths)</p>
−
<h2>Interlabstudy</h2>
+
 
</div>
 
</div>
−
 
  
 
<!-- 13.7. - 19.7. -->
 
<!-- 13.7. - 19.7. -->
 
<div id="dia2" class="dia cal">
 
<div id="dia2" class="dia cal">
 
<h2>Cre-Dronpa</h2>
 
<h2>Cre-Dronpa</h2>
 +
<p>Three part assembly of NLS+Dronpa and Linker 6/9/12/15+Cre_sn with pTUM104</p>
 +
<p>Transformation of L9-Cre, pTUM-L12-Dronpa, pTUM-L15_Dronpa and L15-Cre in yeast</p>
 +
<p>Colony PCR of pTUM-L12_Cre (no successs), pTUM-L9-Cre, pTUM-L12-Dronpa (no successs), pTUM-L15_Dronpa (no successs), pTUM-L6-Dronpa, pTUM-L9-Dronpa (no successs)</p>
 
<h2>Cre Reporter Cassette</h2>
 
<h2>Cre Reporter Cassette</h2>
−
<h2>pRS Vectors</h2>
+
<p>Three part assembly of RFP+tADH,loxP+nluc and pADH+ loxP with pTUM100 (no positive results)</p>
−
<h2>Sensors/Promotors</h2>
+
−
<h2>Interlabstudy</h2>
+
 
</div>
 
</div>
  
  
−
<!-- 20.7. - 26.7. -->
+
<!-- 20.7. - 26.7. Woche 4 -->
 
<div id="dia3" class="dia cal">
 
<div id="dia3" class="dia cal">
 
<h2>Cre-Dronpa</h2>
 
<h2>Cre-Dronpa</h2>
 +
<p>Colony PCR of pTUM-L9-Dronpa, pTUM-L15-Dronpa (no successs), pTUM-L15_Cre</p>
 +
<p>Three part assemblies: NLS+Dronpa, L12+Dronpa with pTUM104; L9+Cre, L15+Cre, L12+Cre with pTUM100</p>
 +
<p>Two part assemblies: L6+Cre_sn, L9+Cre_sn, L12+Cre_sn, L15+Cre_sn, pTUM104+Dronpa, pTUM104-Cre, pTUM104+Cre_sn, </p>
 +
<p>Transformation of all assemblies only successful with pTUM104-L12-Dronpa </p>
 
<h2>Cre Reporter Cassette</h2>
 
<h2>Cre Reporter Cassette</h2>
−
<h2>pRS Vectors</h2>
+
<p>Three part assembly: RFP+tADH, loxP+nluc and pADH+loxP with pTUM100</p>
−
<h2>Sensors/Promotors</h2>
+
<p>Two part assemblies: pTUM104+nluc, pTUM104+luc</p>
−
<h2>Interlabstudy</h2>
+
 
</div>
 
</div>
  
  
−
<!-- 27.7. - 2.8. -->
+
<!-- 27.7. - 2.8. Woche 5-->
 
<div id="dia4" class="dia cal">
 
<div id="dia4" class="dia cal">
 
<h2>Cre-Dronpa</h2>
 
<h2>Cre-Dronpa</h2>
−
<h2>Cre Reporter Cassette</h2>
+
<p>Control restriction and transformation of pTUM100 and pTUM104 for trouble shooting</p>
 +
<p>Two part assemblies:L9-Cre, L15-Cre, L12-Dronpa with pTUM100; three part assembly: Dronpa+NLS with  pTUM104</p>
 +
<p>Colony PCR of pTUM100 and pTUM104 primers</p>
 
<h2>pRS Vectors</h2>
 
<h2>pRS Vectors</h2>
−
<h2>Sensors/Promotors</h2>
+
<p>Mutagenesis and transformation of pRS313 and pRS316 (both not successful) </p>
 
<h2>Interlabstudy</h2>
 
<h2>Interlabstudy</h2>
 +
<p>Transformation of J23101 and J23106 in pSB1C3 </p>
 
</div>
 
</div>
  
  
−
<!-- 3.8. - 9.8. -->
+
<!-- 3.8. - 9.8. Woche 6-->
 
<div id="dia5" class="dia cal">
 
<div id="dia5" class="dia cal">
 
<h2>Cre-Dronpa</h2>
 
<h2>Cre-Dronpa</h2>
 +
<p>Ligation and transformation of assemblies: Cre, Cre_sn, Dronpa with pTUM 104, Dronpa with pSB1C3, L9-Cre-L9-Dronpa, L9-Cre_sn-L9-Dronpa, L12-Cre-L12-Dronpa, L12-Cre_sn-L12-Dronpa</p>
 +
<p>Retransformation of pSB-Dronpa</p>
 +
<p>Colony PCR of pTUM104-Cre, pTUM104-Cre_sn, pTUM104-Dronpa, pSB1C3-Cre_ns, pTUM100-L9-Cre_sn, pTUM100-L15-Cre_sn</p>
 
<h2>Cre Reporter Cassette</h2>
 
<h2>Cre Reporter Cassette</h2>
−
<h2>pRS Vectors</h2>
+
<p>Ligation and transformation of assemblies: RFP-tADH, nluc, luc (not successful) with pTUM104, pADH-loxP, loxP-nluc with pTUM100</p>
−
<h2>Sensors/Promotors</h2>
+
<p>Retransformation of pSB-loxP-WT and pSB-nluc</p>
 +
<p>Colony PCR of pTUM104-RFP-tADH, pTUM104-loxP-nluc, pTUM104-pADH-loxP, pTUM104-nluc, pTUM104-luc</p>
 
<h2>Interlabstudy</h2>
 
<h2>Interlabstudy</h2>
 +
<p>Colony PCR of pSB1C3-J23101, pSB1C3-J23106, pSB1C3-J23101+I13504, pBS1C3-J23106+I13504, pSB1C3-J23117+I13504</p>
 +
<p>Transformation of J23151 and TetR repressible promoter (positive control)</p>
 +
<p>Ligation and transformation of pSB1C3-J23101+I13504 (not successfull), pBS1C3-J23106+I13504(not successfull), pSB1C3-J23117+I13504</p>
 
</div>
 
</div>
  

Revision as of 20:28, 18 September 2015

<
>

Dronpa-Caged Cre

Ligation of gene blocks (Cre, Cre_sn, Dronpa, HCV, HCV+linker, nluc), linker L6, L9, L12, L15, nDegron and NLS with pSB1C3 and transformation in DH10beta:

Colony PCR and test restriction to check for correct transformation

pSB-Cre_sn and pSB-Luciferase did not yield correct colonies (transformation repeated), the other constructs resulted in correct colonies

Cre Reporter Cassette

Ligation of gene blocks (tADH, loxP WT, loxP66, loxP71) with pSB1C3 and transformation in DH10beta

pRS Vectors

Mutagenesis primer design pRS313, pRS315, pRS316

Cre-Dronpa

Colony PCR of pSB-luc

Transformation of pSB-Gal and pSB-luc in DH10beta

Three part assembly of Linker(6/9/12/15) + Cre, Linker(6,9,12,15) + Dronpa with pTUM100 and transformation in yeast resulting in mixed colony PCR results (L6-Cre, L9-Cre, L12-Cre and L9-Dronpa yielded correct bands, the other assamblies did not result in correct fragment lengths)

Cre-Dronpa

Three part assembly of NLS+Dronpa and Linker 6/9/12/15+Cre_sn with pTUM104

Transformation of L9-Cre, pTUM-L12-Dronpa, pTUM-L15_Dronpa and L15-Cre in yeast

Colony PCR of pTUM-L12_Cre (no successs), pTUM-L9-Cre, pTUM-L12-Dronpa (no successs), pTUM-L15_Dronpa (no successs), pTUM-L6-Dronpa, pTUM-L9-Dronpa (no successs)

Cre Reporter Cassette

Three part assembly of RFP+tADH,loxP+nluc and pADH+ loxP with pTUM100 (no positive results)

Cre-Dronpa

Colony PCR of pTUM-L9-Dronpa, pTUM-L15-Dronpa (no successs), pTUM-L15_Cre

Three part assemblies: NLS+Dronpa, L12+Dronpa with pTUM104; L9+Cre, L15+Cre, L12+Cre with pTUM100

Two part assemblies: L6+Cre_sn, L9+Cre_sn, L12+Cre_sn, L15+Cre_sn, pTUM104+Dronpa, pTUM104-Cre, pTUM104+Cre_sn,

Transformation of all assemblies only successful with pTUM104-L12-Dronpa

Cre Reporter Cassette

Three part assembly: RFP+tADH, loxP+nluc and pADH+loxP with pTUM100

Two part assemblies: pTUM104+nluc, pTUM104+luc

Cre-Dronpa

Control restriction and transformation of pTUM100 and pTUM104 for trouble shooting

Two part assemblies:L9-Cre, L15-Cre, L12-Dronpa with pTUM100; three part assembly: Dronpa+NLS with pTUM104

Colony PCR of pTUM100 and pTUM104 primers

pRS Vectors

Mutagenesis and transformation of pRS313 and pRS316 (both not successful)

Interlabstudy

Transformation of J23101 and J23106 in pSB1C3

Cre-Dronpa

Ligation and transformation of assemblies: Cre, Cre_sn, Dronpa with pTUM 104, Dronpa with pSB1C3, L9-Cre-L9-Dronpa, L9-Cre_sn-L9-Dronpa, L12-Cre-L12-Dronpa, L12-Cre_sn-L12-Dronpa

Retransformation of pSB-Dronpa

Colony PCR of pTUM104-Cre, pTUM104-Cre_sn, pTUM104-Dronpa, pSB1C3-Cre_ns, pTUM100-L9-Cre_sn, pTUM100-L15-Cre_sn

Cre Reporter Cassette

Ligation and transformation of assemblies: RFP-tADH, nluc, luc (not successful) with pTUM104, pADH-loxP, loxP-nluc with pTUM100

Retransformation of pSB-loxP-WT and pSB-nluc

Colony PCR of pTUM104-RFP-tADH, pTUM104-loxP-nluc, pTUM104-pADH-loxP, pTUM104-nluc, pTUM104-luc

Interlabstudy

Colony PCR of pSB1C3-J23101, pSB1C3-J23106, pSB1C3-J23101+I13504, pBS1C3-J23106+I13504, pSB1C3-J23117+I13504

Transformation of J23151 and TetR repressible promoter (positive control)

Ligation and transformation of pSB1C3-J23101+I13504 (not successfull), pBS1C3-J23106+I13504(not successfull), pSB1C3-J23117+I13504

Cre-Dronpa

Cre Reporter Cassette

pRS Vectors

Sensors/Promotors

Interlabstudy

Cre-Dronpa

Cre Reporter Cassette

pRS Vectors

Sensors/Promotors

Interlabstudy

Cre-Dronpa

Cre Reporter Cassette

pRS Vectors

Sensors/Promotors

Interlabstudy

Cre-Dronpa

Cre Reporter Cassette

pRS Vectors

Sensors/Promotors

Interlabstudy

Cre-Dronpa

Cre Reporter Cassette

pRS Vectors

Sensors/Promotors

Interlabstudy

Cre-Dronpa

Cre Reporter Cassette

pRS Vectors

Sensors/Promotors

Interlabstudy