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==Transformation== #Thaw one 50ul vial of One Shot® Competent Cells on ice. #Add 2 μl ligation product into one vial of One Shot® Chemically Competent E. coli and mix by stirring gently with the pipette tip. Store the rest of the ligation products in -20 C. Do not mix by pipetting up and down. #Incubate on ice for 30 minutes. Note: Longer incubations on ice do not seem to have any affect on transformation efficiency. #Heat-shock the cells for 30 seconds at 42 °C without shaking. #Immediately transfer the tubes to ice for 2 minutes. #Add 250 μl of room temperature S.O.C. medium. #Cap the tube tightly and shake the tube horizontally (256 rpm) at 37°C for 1 hour. #Spread 20 μl from each transformation on a prewarmed selective plate and incubate overnight at 37°C. (To ensure even spreading of small volumes, add 20 μl of S.O.C. medium. The manual recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies.)
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