Team:Stockholm/20 July 2010




flourecent pEX

over expression

  • Take an aliquote of the ON culture and add fresh culture (1:100)
  • Also, 4 small colonies were taken from Johan's bFGF as a control
  • Meassure after ~2h and then more often until OD=0.6
  • When OD=0.6 add IPTG to induce protein production. Take 1ml sample after 0h, 2h and 3h
  • Centrifuge down the cells (13000rpm, 1m) trash LB, add 50µl H2O and resuspend the cells
  • Add 50µl ROSB to denature the proteins
Freeze samples

Site-directed mutagenesis

Designing primers

  • Using Agilent Technologies primer design software
  • Restriction site Age1 A|CCG GT --> A CCA GT easy changed, commonly used codon
Pst1 C TGC A|G --> C TGA A|G best possible, though pyridin/pyrimidin change

The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/