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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

Ligation

(estimated time: 20 min + 12-16 hours overnight incubation)

(For every ligation) Add 50 ng of vector Add       Heat DNA mix at 65°C for 5 min for DNA denaturation Add 1 µl of T4 Ligase buffer Add 1 µl of T4 Ligase 10 µl final volume Incubate at 16°C overnight Then, ligation can be conserved at 4°C or can be transformed Before transformation you have to inactivate T4 Ligase: Heat ligation at 65°C for 10 min.

Roche T4 Ligase 10X Roche T4 Ligase Buffer ddH2O

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