Methods and Materials

This experiment is a three day experiment. On day one, prepare the agar plates and the E.coli cells plates( which will be incubated over night in an incubation oven at 37C). When preparing the agar plates, the two striped plates will not contain ampicillin and the two unstriped plates will contain ampicillin. put the CaCl2 solution and the pFluoroGreen on ice (They should be keep in the ice so it stay coold at all times). On day two of the lab, transform cells and plate for overnight incubation. Take two micro centrifuge, label one “DNA+” and the other one “DNA-”(this will contain no pFG plasmid). Use a sterile 1ml pipette, add 250m l(0.25ml) of ice cold CaCl2 solution to each tube. Transfer 2-4 colonies of E.coli from the source plate to each test tube using a toothpick. Be careful do not scrape up any agar when taking the E. coli from the source plate. Do not wipe the bacteria cells on the side of the micro centrifuge. Twist the toothpick vigorously and up and down in the cold CaCl2 to dislodge the cells. To prevent cross-contaminations use different toothpick for each test tube. Then close the tubes and tap or shake the tubes to suspend the cells completely. Add 10m l of pFG (pFluoroGreen) to the “DNA+” test tube. Incubate the tow test tubes on ice fro fifteen minutes in a small beaker. Transfer both test tube to a 42° C water bath for 90seconds. Then return both tubes immediately to the ice bucket and incubate for 2 minutes. Use a clean sterile 1ml pipette and add 250m l of Luria recovery Broth to each test tube and mix by shaking. Incubate both test tubes for 30 minutes in a 37° C water bath fro a recovery period. To make sure that the temperature is correct, put a thermometer in each water bath. While the tubes are incubating label the agar plates. Label one striped plate as “LB-”, and the other striped plate as “LB+”. Labe one unstriped plate as “LB/Amp-” and the other unstriped plate as “LB/Amp+”. After the recovery period, remove the tubes from the water bath and place them on the lab bench. Then proceed on to plating the cells fro incubation. Use a sterile 1ml pipette to transfer 250m l of recovered cells from the “DNA-” tube to the middle of the “LB-” and “LB/Amp-” plates. Spread the cells over the entire plate with a sterile inoculating loop. First spread the cell in one direction then spread the cells again in 90° of the first direction. Cover both plates and allow the liquid to be absorbed(approximately 15-20 minutes). Use another 1ml pipette to transfer 250m l of recovered cells from the “DNA+” tube to the middle of the “LB+” and the “LB/Amp+” plates. To avoid contamination when plating, do not set the lid down on the lab bench, lift the lid of the plate only enough to allow spreading. Use a different loop to spread recovered cells on each plate. Be careful to avoid gouging the loop into the agar. Stack the plates on top of each other and tape them together. Put your initial son the tapped set. After the cell suspension is absorbed by the agar which takes around 20 minutes, flap the stake of plates over so that the agar side is on the top. Then place it into a 37° C bacterial incubation oven fro overnight incubation. On day three , observe transform ants and controls. Darken the room and use a long wave U.V light to visualize the transformed cells that will glow as green due to the expression of the green fluorescent protein. Count the colonies of transformed E.coli in each plate. Dispose all material porperly in a red hazard bag, this includes the bacteria plates. Compare result with other groups.