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From 2010.igem.org

Contents 1 Mimmi 1.1 flourecent pEX 1.1.1 over expression 1.2 Site-directed mutagenesis 1.2.1 Designing primers

Mimmi

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}