Team:Stockholm/13 July 2010
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[[Image:Coommasie_Johan_bFGF_7july_13july.jpg]] | [[Image:Coommasie_Johan_bFGF_7july_13july.jpg]] | ||
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+ | 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. | ||
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+ | 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 === | === Site-directed mutagenesis === | ||
- | * Performed site-directed mutagenesis on bFGF from received vector, and left with Dpn I overnight. | + | * Performed site-directed mutagenesis on bFGF from received vector, and left with Dpn I overnight. ([https://2010.igem.org/Team:Stockholm/Protocols#Site-directed_mutagenesis protocol]) |
* Forward primer | * Forward primer |
Revision as of 22:35, 14 July 2010
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