| 2. Transformations: The ligation products from August 5th were transformed into TOP10 competent cells. 5uL of ligation product was combined with 50uL of thawed cells, and this mixture was incubated on ice for 30 min. Cells were heat shocked at 42 C for 30 seconds, incubated on ice for 5 min, and then 250 uL prewarmed SOC was added to each. Cells were allowed to recover at 37 C with shaking at 225 RPM for 1 hour, and then 125uL of each was spread on LB plates with kanamycin for selection and allowed to grow overnight.
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