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== '''Colony Prep''' == *Purify with minelute [[File:10-21-11 gel.jpeg|400px]] '''Ligation''' *I selected for 300-600 bp, so the mean size is ~450 bp. This corresponds to 32 ng DNA/rxn *Create the following: 32 ng DNA, 1 uL buffer, 1 uL vector, 1 uL t4 ligase, fill to 10 uL with water *Incubate at 16 C for 1 hr '''Transfection''' *Add 1 uL of ligated product to thawed competent cells *Incubate on ice for 30 min *Heat shock at 42 C for 30 s *Put back on ice for 2 min *Add 250 uL SOC media to each and incubate at 37 C for 1 hr with shaking *Add 20 uL XGAL to each plate (warm up plates and XGAL to rt) and incubate in dark for 10 min *Add 50 uL cells to each plate and incubate overnight at 37 C
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