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From 2010.igem.org

Johan

Visualized coommasie gel

(7 July)

Lane 1: buffer, 2-5: Nina's IgG Protease (0h, 1h, 2h, 3h after IPTG induction), 6-9: my bFGF (0h, 1h, 2h, 3h after IPTG induction), 10: ladder.

bFGF is fused to GST in the vector that was used, with a total size of ~40 kDa, thus showing the correct size on the gel.

Site-directed mutagenesis

• Performed site-directed mutagenesis on bFGF from received vector, and left with Dpn I overnight. (protocol)

• Forward primer

Mol weight (g/mol): 14784,77

Concentration (pmol/µl): 163,08

163E-12 mol/µl * 14789 g/mol = 2,4 µg/µl

To get 125 ng/µl: 2,4E-6/125E-9 = 19

A small volume was diluted 19x and 1 µl was used in the PCR

• Reverse primer

Mol weight (g/mol): 14743,71

Concentration (pmol/µl): 119,37

119E-12 mol/µl * 14743 g/mol = 1,75 µg/µl

To get 125 ng/µl: 1,76E-6/125E-9 = 14

A small volume was diluted 14x and 1 µl was used in the PCR

• dsDNA Template

Concentration: ~300 ng/µl.

A small volume was diluted 30x and 1 µl was used in the PCR