Recombinant DNA Bacterial Transformation

Student InstructionsMid-Log Culture Prep

Recombinant TransformationDay 1

Overnight Culture Plate

Using the extra “LB” agar plate, streak the MM 294 E.coli culture on the plate and incubate 18-24 hours.

Recombinant TransformationDay 2

Mid-Log Culture

Label a sterile 50 ml conical tube with your name and date.

Recombinant TransformationMid-Log Culture

Use a sterile pipette to transfer one pipette full (5 ml) of sterile LB broth into the conical tube.

Recombinant TransformationMid-Log Culture

Locate a well-formed colony from your plate of bacteria grown the day before. Use a sterile inoculating loop, bent paper clip, or toothpick to scrape up the colony and transfer it to the LB broth in the conical tube. Your instructor may have started the culture several hours before class.

Recombinant TransformationMid-Log Culture

Alternatively: Incubate 4 hours in the incubator with occasional shaking by hand. For best results, shake the tube a couple of times an hour when possible. After the 4 hour incubation period, immediately proceed to the “Making Competent Cells” protocol.

Incubate the tube for 3-4 in a shaking water bath or use an orbital shaker in an incubator.