E. Colo Lab Report

METHODS
In this experiment, plasmid lux and a control plasmid (pUC18) will be introduced into E. coli by transformation. There are four basic steps to the procedure;
Preparation of competent cells
(These steps should be performed by the instructor.)
1. Place a vial of CaCl2 solution and the tube of E. coli in the ice bath.
2. Using a sterile pipet, transfer 590µL CaCl2 solution to the tube containing 50µL of the bacteria.
3. Tap the vial with the tip of your index finger to mix the solution.
4. Incubate the cells for at least 10 minutes on ice. The cells are then called competent because they can take up DNA from the medium. If desired, the cells can be stored in the CaCl2 solution for 12–24 hours. B. Uptake of DNA by competent cells
1. Label
Place both tubes in an ice bath.
3. Using a sterile micropipette, add 5µL control plasmid to the tube labeled “C” and 5µL plasmid lux to the tube labeled “lux”. Make sure to keep all tubes in the ice until instructed otherwise. (Concentration=0.005µg/µl).
4. Gently tap the tube of competent cells with the tip of your index finger to ensure that the cells are in suspension.
5. using a sterile transfer pipette add 70µL of the competent cells to each of the 2 tubes.
6. Tap each of these tubes with the tip of your index finger to mix these solutions, and store both tubes on ice for 15 minutes. Obtain one additional tube. Add 35µL of the competent cell to each tube and label the tubes “NP” (no plasmid).
7. HEAT SHOCK; Transfer all the tubes to a water bath preheated to 37˚C, and allow them to sit in the water bath for 5
Obtain agar plates from your instructor and label them as in the figure 1 provided below.
2. Using a sterile pipet, remove 130µL mixed bacterial suspension from the tube “C “, remove the lid from the control plate and dispense the bacteria unto the agar. Use a cell spreader to spread the bacteria evenly onto the agar surface. Use of the cell spreader;
Dip the cell spreader into ethanol. Pass the spreader across the flame of ethanol lamp. Make sure you only pass it through the flame and not keep it in the flame. Once the ethanol has burnt, keep the spreader still for about 30 seconds. This allows the spreader to cool down before you start spreading the cells. Once the spreader has cooled down, use the spreader to evenly distribute the cells suspension over the entire surface of the plate. Return the cell spreader to the ethanol contained without flaming and repeat the whole procedure until you have depleted all the bacteria.
3. Transfer 130µL bacterial suspension from the “lux” tube to the “lux” plate and spread these cells onto the agar surface as described in the previous step.
4. Cells from the two tubes that did not contain plasmids (NP) should be plated onto two plates
(NP) as described in steps