Team:Groningen/Notebook/9 July 2009

GVP Cluster
Inoculation of TY-medium for Glycerol Stocks and Plasmid isolation


 * BBa_J23109 (labelled as no.1) vector BBa_J61002 in E.coli TOP10
 * BBa_J23100 (labelled as no.2) vector BBa_J61002 in E.coli TOP10 (red colony)
 * BBa_J23106 (labelled as no.3) vector BBa_J61002 in E.coli TOP10 (red colony)
 * The three plates showing new colonies were used to inoculate 5mL TY-medium with amp. (final conc. 100mg/ul).
 * The tubes were put in a waterbath at 37C (200 rpm) and grown o.n.

Transporters
[[Image:HmtA_SDS_gel.jpg|200px|thumb|right|[Team:Groningen/Team|First results!]] Purification of HmtA (PA Q9I147 6xHis Tag)
 * SDS-gel electrophosis
 * Make a 12% SDS-gel
 * Fill gel with samples Wash 1-2 20mM imidazole, Wash1-2 40mM imidazole, Elute1-3 and ladders
 * Run 2h at 100V
 * Stain the gel in Coomassie blue (2h)
 * Destain with demiwater (2x, 10 min microwave)

Vectors
Measure concentration with Nanodrop

{|

Dry
A lot of time was spent on finding dissociation constants and figuring out how ArsR/D work, and thinking of how to model their interaction. An input function was derived for the ArsR/D operator, but it is not yet completely clear how this should be used in a model (it is a function of the ArsR/D concentration, but at the same time produces ArsR/ArsD). It would probably be better to start with As(III) and derive an input function from that, if at all possible.