User:Michael Verhoeven/Notebook/iGEM 2009 M. Verhoeven Parts/2009/07/07

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Labwork
Plating of E.coli TOP10 cells

The plates with transformed E.coli TOP10 cells contained between 5-50 colonies for the non-diluted plates, depending on which plasmid was transformed into the cells. To increase the number and size of the colonies, the plates were stored at 37°C for an additional 24 hours.

Plasmid Purification

Plasmid isolation was performed on the cultures of GVP and Terminator containing cells with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".
 * From each tube 2mL of culture was collected in a 2mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
 * Cells were resuspended in 200μL Resuspension Solution by up and down pipetting.
 * To the mixture 200μL Lysis Solution was added, mixed by inverting the cup, and stored at room temperature for 5 minutes.
 * 350μL of Neutralisation Solution was added and the tubes inverted.
 * Cell debri was pelleted by centrifugation at full speed for 1 min.

Concentration of Plasmids

The concentration of isolated plasmid was determined with the use of a nano-drop.

GVP eluted in MQ
 * 88.2 ng/μL
 * 1.83 (260/280)
 * 2.08 (260/230)

GVP eluted in elution buffer
 * 92.2 ng/μL
 * 1.91 (260/280)
 * 2.78 (260/230)

Terminator eluted in MQ
 * 35.3 ng/μL
 * 1.74 (260/280)
 * 1.97 (260/230)

Terminator eluted in elution buffer
 * 38.8 ng/μL
 * 1.89 (260/280)
 * 1.93 (260/230)

Restriction analysis

The plasmids containing GVP were cut with EcoRI and XbaI fast digest enzymes. The double digestion should result in two fragments of 1400 and 8000 bp in size.

The cups were incubated in a heat block at 37°C for 15 min. followed by adding 4μL 6x loading buffer for gel electroforese.
 * 6μL MQ
 * 10μL plasmid in MQ
 * 2μL Fast digest buffer
 * 1μL EcoRI fast digest enzyme
 * 1μL XbaI fast digest enzyme

Gel electroforese

10μL of each sample was loaded on a 2% agarose gel with EtBr and a 1kb ladder was used (see picture).