Aim 1: DNA reproduction+PCR
For PCR key see PCR key
Colourcode for the primers: Annealing part, mutagenised part, other sequences integrated into primer
PCR1: replication of the Tet-inducible CMV promotor [X]
Primer used: 1TreF 2TreR
expected product size: 492 bp
PCR4a: replication human bak with mutagenesis (Pst1) [ ]
Primer used: 7BakF 8BakMutPR
expected product size: 330 bp
PCR4b: replication human bak with mutagenesis (Pst1) [ ]
Primer used: 9BakMutPF 10BakR
expected product size: 376 bp
PCR5: joining PCR of human bak [ ]
Primer used: 7BakF 10BakR
expected product size: 688 bp
PCR6: replication of the SV40-polyadenylation site [X]
Primer used: 11PAF 12PAR
expected product size: 237 bp
PCR9: replication of eGFP with attP in primer [ ]
Primer used: 20eGFPattPF 21eGFPR
expected product size: 808 bp
PCR10: Replikation of PhiC31o [X]
Primer used: 22PC31oF 23PC31oR
expected product size: 1888 bp
Note: for attB two Primers ( 18attBF 19attBR) used.
Aim 2: Inserting PCR Products in pSB1C3 and verifying Sequenz
PCR1: Insertion [ ] -> Sequenz of PCR1 from sequenzing: confirmed [ ]
PCR5: Insertion [ ] -> Sequenz of PCR5 from sequenzing: confirmed [ ]
PCR6: Insertion [ ] -> Sequenz of PCR6 from sequenzing: confirmed [ ]
PCR9: Insertion [ ] -> Sequenz of PCR9 from sequenzing: confirmed [ ]
PCR10: Insertion [ ] -> Sequenz of PCR10 from sequenzing: confirmed [ ]
Aim 3: Assembling Biobricks
We are using the 3A System to assemble Biobricks.
Assembling Construct 1
Assembling Construct 2
Assembling Construct 3
Aim 4: Testing products
Construct 1
- Transform into HeLa cells to see if they survive.
-> Check the leakiness of tet-on-promoter
- Induce tet-on-promoter to see, if cells die.
-> Check if construct 1 is working
Construct 2
- Sequencing
Construct 3
- Transform into HeLa cells
- Western Blot or Proteinchromatography
-> Check if PhiC31o is read off
Aim 5: Testing system
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