Team:LMU-Munich/Jump-or-die/Schedule

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 [ ]

Primer used: 22PC31oF 23PC31oR expected product size: 1888 bp

Note: for attB two Primers ( 18attBF 19attBR) used.

Aim 2: Assembling Biobricks

We are using the 3A System to assemble Biobricks.

Assembling Construct 1

Assembling Construct 2

Assembling Construct 3

Aim 3: 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

- transform into HeLa cells and see if eGFP is being translated

-> Check if CMV-promoter is working and construct is being read-off completely

- Western Blot FALSEEE

Aim 4: Testing system