1. Recombinant DNA Bacterial Transformation Student Instructions Mid-Log Culture Prep

2. Recombinant Transformation Day 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.

3. Recombinant Transformation Day 2 Mid-Log Culture Label a sterile 50 ml conical tube with your name and date.

4. Recombinant Transformation Mid-Log Culture Use a sterile pipette to transfer one pipette full (5 ml) of sterile LB broth into the conical tube.

5. Recombinant Transformation Mid-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.

6. Recombinant Transformation Mid-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.