Team:Groningen/Notebook/11 August 2009

From 2009.igem.org

Wet

GVP Cluster

→ DONE isolate plasmids from overnight precultures

→ DONE plate on cultures for storage in fridge (amp)

→ TODO cut plasmid and GVP containing plasmid for ligation (ligation tomorrow)

→ TODO purify wanted fragments (tomorrow)

→ DONE make o.n. precultures from plate colonies

www.sigmaaldrich.com

Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 J61002] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 medium] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 low] constitutive promoters with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html 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 20μL MQ and stored in the fridge

Concentrations

→ These plasmids come from the glycerol stocks contaning E.coli TOP10 cells with iGEM plasmid [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 J61002] and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 medium] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 low] constitutive promoters with RFP as reporter gene behind them.

→ The GVP cluster can be ligated behind the promoters and self ligation of RFP will turn unwanted colonies red!

Plates

LB-agar plates with 50 μg/mL ampicillin were freshly prepared, and one drop of each o.n. preculture was spotted and subsequently swiped three times with sterile needle. The plates were stored at 37°C overnight.

Precultures

Over night cultures in 5mL LB-amp₅₀ medium were prepared from the following colonies:

→ E.coli TOP10 pSB1AC3-med. const. promoter - GVP (amp.) (4x) (1 to 4)

→ E.coli TOP10 pSB1AC3-low const. promoter - GVP (amp.) (4x) (5 to 8)

and put in the 37°C waterbath at 200 rpm.

Transporters

Metal Accumulation

Vectors

Dry