Bacterial Transformation

Bacterial Transformation and its Process

Step #1: You need to label one of the closed test tubes +pGLO and another -pGLO, then place it in the foam tube holder.
Step #2: Open the tubes and use a pipet to transfer 250ul of the CaCl2. Place this tube on ice.
Step #3: Use the sterile loop to pick up a single form of bacteria from the starter plate. Get the +pGLO tube ready, then immerse the loop into the transformation solution at the bottom of the tube. Spin the loop until the entire colony is dispersed. Place the tube back in the ice bucket. Repeat with -pGLO with a new sterile loop.
Step #4: Examine the plasmid DNA with the UV light. Immerse the new plasmid into the DNA stocktube. Withdraw another loop of bacteria. In this step there should be a film of plasmid across the ring. Mix the loopful into the cell suspension of the +pGLO tube. Once again put it back on the ice. DO NOT add plasmid DNA to the -pGLO tube.
Step #5: Incubate the tubes that are on ice for at the most 10 minutes. The bottoms of the tubes should be in contact with the ice.
Step #6: Next label four agar plates as follows: LB/amp plate label +pGLO; LB/amp/ara plate label -pGLO; LB/amp label -pGLO; and the LB plate -pGLO.
Step #7: After that step transfer both tubes into the water bath that's set at 42 degrees Celsius for exactly 50 seconds.
Step #8: Place the tubes in ice for two minutes then take them off the ice and open the tube. Use a new pipet to add 250 ul of LB nutrient broth to the tube and reclose it. Repeat with a new pipet to the other tube. Incubate the tubes at room temp for 10 minutes.
Step #9: Mix the tubes softly. Use a new sterile pipet for each tube, pipet 100ul of the transformation control suspensions to appropriate plates.
Step #10: Spread the suspensions evenly with a sterile loop.
Step #11: Stack the plates and tape them together, upside down. Place it in the Biology incubator overnight which is set around 37 degrees Celsius.

Observations:

+pGLO; LB/amp: has the gene but it isn't "turned on". It has a medium to heavy population which is white and yellow. Our estimated count was 130. Transformation plates

+pGLO; LB/amp/ara: has the protein that is turned on, which makes it glow. It has a medium to heavy population that's yellow then a light white. Our estimated count is 149. Transformation plates

-pGLO; LB/amp: has no population and no bacteria!! Control Plates

-pGLO; LB: has a HEAVY bacterial lawn!!! Control Plates

Overall Analysis:

Plasmid seemingly does not make a difference to the health of the illness the bacteria may cause.

The amp kills the bacteria unless a gene from the plasmid is LB/amp +/-.