IMPORTANT: In addition to wearing the appropriate personal protective equipment, be sure to take care to keep your face away from suspension cultures, and to avoid inhaling reagents. Do not touch your face while performing the experiment. Always wash your hands before and after every experiment.
When you are ready to safely begin, check to make sure you have a tube or tubes of competent E. coli cells in your ice bucket.
Then, transfer 50 µL from one of these tubes to each of two new micro-centrifuge tubes, one labeled ‘–’, and one labeled ‘+’.
Next, pipet 2 µL of p-GREEN plasmid into the plus tube and flick the tube gently to incorporate the plasmid throughout the bacterial solution. HYPOTHESES: The experimental hypothesis is that on plates containing no antibiotic, bacteria with or without the plasmid can survive, and so both will grow lawns of bacteria. Conversely, on the plates containing the ampicillin antibiotic, only the bacteria successfully transformed with the resistance plasmid will grow, whereas the minus plasmid plates will contain no bacteria. The null hypothesis of this experiment is that there will be no difference in bacterial growth between the plates.
Incubate both tubes on ice.
After 30 minutes submerge both tubes 3/4 of the way into the water of a 42 °C water bath for 30 seconds.
At the end of the water bath incubation, place the tubes back on ice for 5minutes, then add 950 μL of room temperature SOC media to each tube.
Incubate the tubes in a shaker at 37 °C for 60 minutes at 250 rpm.
While the tubes are incubating, label the bottoms of one LB and one LB/ampicillin, or AMP plate, “+ plasmid”, and the bottoms of one LB plate and one LB/AMP plate “- plasmid”.
At the end of the incubation, pipet 50 μL from the “+ plasmid” cell tube onto both the LB/AMP plus plate and the LB plus plate.
Then, with a fresh tip, add 50 μL of the bacteria that were not exposed to the plasmid to each of the minus plates.
Then, spread each plate using a fresh pipette tip.
Cover the plates with their lids, and then seal them with laboratory film.
Then store the plates upside down in a 37 °C incubator for 24 hours.