Team:Groningen/Notebook/17 September 2009

From 2009.igem.org

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Wet

GVP Cluster

Planning

TODO work out the wiki page for GVP
DONE make a doodle for presentation planning (1-19 oct.)
TODO media attention
DONE place an ethics survey link on twitter


TODO clone pArsR-GVP into pSB2K3
TODO clone repeat out of GVP cluster
TODO make glycerol stocks of constructs
TODO enter info on part registry


Cultures

The overnight cultures with LB-amp100 medium of colonies E.coli TOP10 with pNL29 (3x), and pArsR-GVP (1-3) all showed expected growth of bacteria.


www.sigmaaldrich.com

Plasmid isolation

Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".

  • From each tube 4mL of culture (8mL for pNL29) was collected in a 2.0mL cup (tubes from pArsR-GVP, GVP and pNL29 were combined), and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
  • Plasmids were eluted with 30μL MQ and stored in the fridge


Concentrations

Plasmid Conc. ng/μL 260/280 260/230 -20 box (michael Restriction Control
pArsR-GVP (pSB2K3, no.1) 40.6 1.89 1.77 x Yes (EcoRI/PstI)
pArsR-GVP (pSB2K3, no.2) 47.3 1.87 2.01 x Yes (EcoRI/PstI)
pArsR-GVP (pSB2K3, no.3) 40.4 1.89 1.64 x Yes (EcoRI/PstI)
pNL29 26.4 2.10 1.64 x Yes (MvaI/XhoI)


Restriction for Control and Assembly

The plasmids from the o.n. precultures of pArsR-GVP and pNL29 were cut with PstI/EcoRI and MvaI/XhoI to cut out the entire part between the pre- and suffix and wanted fragment.

Plasmid Amount μL MQ μL Fast digest buffer EcoRI fast digest enzyme PstI fast digest enzyme
pArsR-GVP no.1 16.0 x 3.0 1.0 1.0
pArsR-GVP no.2 16.0 x 3.0 1.0 1.0
pArsR-GVP no.2 16.0 x 3.0 1.0 1.0
Plasmid Amount μL MQ μL R-digest buffer MvaI digest enzyme XhoI digest enzyme
pNL29 16.0 x 3.0 1.0 1.0


340px Generulers 1kb marker Fermentas.jpg

→ From left to right: 1kB ladder, pNL29 (orange dye), pNL29 (blue dye), pArsR-GVP no.1, pArsR-GVP no.2, pArsR-GVP no.3

Transporters

Metal Accumulation

Vectors

Dry

April
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May
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June
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July
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August
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31
September
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October
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November
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