Team:Groningen/Notebook/22 August 2009

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Wet

GVP Cluster

DONE Isolate plasmids from E.coli Top10 BBa_J23101 cells
DONE Plate E.coli Top10 BBa_J23101 cells from o.n. culture
TODO Pellet remaining cells for short storage in -20 box (not needed)
DONE Place the four test cultures with and without GVP on table at room temperature (make foto)
TODO Look at plates stored at 37C with BBa_J61002-pArsR+/pZntR+/pCueO+ and use colonies to grow o.n. cultures for plasmid isolation


Plates

Showed single colony growth on plates with J61002-pMEtal+RBS-RFP plasmids, and were stored in the fridge for future preculture growth.

→ The plates with high and low concentration of transformed cells showed colonies in the expected ratio.
→ On the plates for pZntR+ and pCueO+ no colonies were dark red, indication of ligation of the original high constitutive promoter back into the J61002 vector, or uncut plasmid which was transformed.
→ The plates with pArsR+ plasmid showed only a few non-coloured colonies, mostly medium red colour, andn a few dark red colonies. It was already thought that the pArsR is a bit leaky and causes expression of RFP.

Over Night Cultures

→ Both cultures of BBa_J61002-J23101 showed growth, and can be used for plasmid isolation.
www.sigmaaldrich.com

Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids pSB3K3 with high, medium and low constitutive promoters and GVP with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".

  • From each tube 4mL of culture was collected in a 2.0mL cup, 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

Transporters

Metal Accumulation

Vectors

Dry

April
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July
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August
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September
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October
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November
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