User:Michael Verhoeven/Notebook/iGEM 2009 M. Verhoeven Parts/2009/07/07
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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 isolation was performed on the cultures of GVP and Terminator containing cells with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".
Concentration of Plasmids
The concentration of isolated plasmid was determined with the use of a nano-drop.
GVP eluted in MQ
GVP eluted in elution buffer
Terminator eluted in MQ
Terminator eluted in elution buffer
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.
10μL of each sample was loaded on a 2% agarose gel with EtBr and a 1kb ladder was used (see picture).