Revision as of 17:29, 24 October 2010 by Jara (Talk | contribs)


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] PCR1.jpg

Primer used: 1TreF 2TreR expected product size: 492 bp

PCR4a: replication human bak with mutagenesis (Pst1) [ ] PCR4a.jpg

Primer used: 7BakF 8BakMutPR expected product size: 330 bp

PCR4b: replication human bak with mutagenesis (Pst1) [ ] PCR4b.jpg

Primer used: 9BakMutPF 10BakR expected product size: 376 bp

PCR5: joining PCR of human bak [ ] PCR5.jpg

Primer used: 7BakF 10BakR expected product size: 688 bp

PCR6: replication of the SV40-polyadenylation site [X] PCR6.jpg

Primer used: 11PAF 12PAR expected product size: 237 bp

PCR9: replication of eGFP with attP in primer [ ] PCR9.jpg

Primer used: 20eGFPattPF 21eGFPR expected product size: 808 bp

PCR10: Replikation of PhiC31o [X] PCR10.jpg

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 Sequence

PCR1: Insertion [ ] -> Sequence of PCR1 from sequencing: confirmed [ ]

PCR5: Insertion [ ] -> Sequence of PCR5 from sequencing: confirmed [ ]

PCR6: Insertion [ ] -> Sequence of PCR6 from sequencing: confirmed [ ]

PCR9: Insertion [ ] -> Sequence of PCR9 from sequencing: confirmed [ ]

PCR10: Insertion [ ] -> Sequence of PCR10 from sequencing: confirmed [ ]

Aim 3: Assembling Biobricks

We are using the 3A System to assemble Biobricks.

Assembly of BioBricks:

Assembling Construct 1


Assembling Construct 2


Assembling Construct 3


Aim 4: Testing products

Construct 1

Jump 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

Jump construct 2

- Sequencing

Construct 3

Jump construct 3

- Transform into HeLa cells

- Western Blot or Proteinchromatography

-> Check if PhiC31o is read off

Aim 5: Testing system