Team:Tuebingen/Notebook
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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