Transformation Protocol in DH5α

Thaw an aliquot of competent bacteria on ice.

Add 50 µl of DH5α competent cells gently in a sterile 15 ml polypropylene tube. Add 1 µl of ligation mixture or Gibson Assembly reaction (1 – 10 ng of DNA) to the polypropylene tube

Incubate on ice for 30 minutes.

Heat shock for 45 seconds in a water bath at 42°C, then quickly back on ice for 2 minutes.

Add 500 ul of room temperature SOC medium (Work sterile!!).

Incubate 1 hour at 37°C for antibiotic resistance expression, while shaking (225 rpm).

Spread 1/10 and 9/10 on LB plates (containing the right antibiotic).

Notes: Do not shake the polypropylene tubes during the heat shock! To increase the yield, centrifuge gently (at low speed), remove the excess of medium and then spread on the plates. To determine transformation efficiency