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From 2009.igem.org

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	LB medium DNA resuspension from iGEM 2009 plates Transformation X-Gal staining protocol for beta galactosidase (blue/white screening) Glycerol stocks Plasmid extraction BioBrick digestion with restriction enzymes Electrophoresis DNA gel extraction DNA precipitation with sodium acetate Ligation PCR Sensing ethanol concentration: our do-it-yourself kit

BioBrick digestion with restriction enzymes

(estimated time: 3 hours)

To open vectors: a volume containing 1 µg of purified plasmid 2 µl of buffer H 1 µl of first enzyme 1 µl of second enzyme 20 µl final volume incubate at 37°C for 3 hours To excide fragments: 20 µl of purified plasmid when <7 µg have been extracted. A volume containing 7 µg otherwise. 2.5 µl of buffer H 1 µl of first enzyme 1 µl of second enzyme 25 µl final volume incubate at 37°C for 2 hours and 30 minutes

Roche restriction enzymes Roche buffer H Pre-warmed at 37°C bath Cut and gel-extracted vector ddH2O

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