Genetic Engineering: Bacterial Transformation Lab

Transformation Lab

Procedure

1. Level one closed micro test tube +pGLO and another -pGLO. Label both tubes with your group's name. Place them in the foam tube rack.
2. Open the tubes and using a sterile transfer pipet, transfer 250us of transformation solution (CaCi2).
3. Place the tubes on ice.
4. Use a sterile loop to pick up a single colony of bacteria from you starter plate. Pick up the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile look. repeat for the +pGLO
5. Examine the pGLO plasmid DNA solution with the UV lamp. Note your observations. Immerse a new sterile loop into the plasmid DNA stock tube. Withdraw a lapful. There should be a film of plasmid solutions a cross the ring. This is similar to seeing a soapy film across a ring for blowing soap bubbles. Mix the lapful into the cell suspension of the +pGLO tube. close the tube and return it to the rack on ice. Also close the -pGLO tube. Why not?
6. Incubate the tubes on ice for 10 minutes. Make surety push the tubes all the way down in the rock so the bottom of the tubes stick out and make contact with ice. 
7. While the tubes are sitting on ice, label your four agar plates on the bottom as follows: Label one LB/amp plate:+pGLO Label the LB/amp/ara plate: +pGLO; Label the LB plate: _pGLO.
8. Heat shock. Using the foam rack as a holder, transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42 degrees Celsius, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the warm water. When the 50 seconds are done, place both tubes back on ice. For the best transformation results, the change from the ice (0 degrees Celsius) to 42 degrees Celsius  and then back to the ice must be rapid. Incubate tubes on ice for 2 minutes.
9. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and using a new sterile pipet, add 250 ul of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 minutes at room temperature.
10. Tap the closed tubes with your finger to mix. Using a new sterile pipe for each tube, pipet 100 ul of the transformation and control suspensions onto the appropriate plates.
11. Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of agar by quickly skating the flat surface of a new sterile loop back and for across the plate surface.
12. Stack up your plates and tape them together. Put your group name and class period on the bottom of the stack and place the stack upside down in the 37 degree Celsius incubator until the next day.

Observation
So in the end we had plates that we observed.  None of the bacteria grew but one of them was killed. The color of the bacteria was neon green.