Bacterial Transformation

 I tried to transform Agrobacteria and Escherichia coli (E. coli) with plasmid DNA . Bacterial Transformation is the “process by which the genetic material carried by an individual cell is altered by the incorporation of foreign (exogenous) DNA into its genome” (MedicineNet.com, “Definition of Genetic transformation”).

At first, I used a micropipette to transfer 250 ul of transformation solution (CaCl₂) to the tube labeled + pGLO and another 250 ul to the tube labeled - pGLO. Then, I placed the tubes in a container of crushed ice.

I used a sterile loop to pick up several colonies of my bacteria (Agrobacteria and E.Coli) from the starter plates and transferred the bacteria to the + pGLO tube by spinning the loop rapidly after it is immersed in the liquid. I repeated this procedure by transferring several colonies of bacteria to the - pGLO tube.

I immersed a sterile loop into the bottle containing plasmid DNA. When the center of the loop was coated with a soap-like film, transferred it the + pGLO microtube .The - pGLO microtube will not receive any plasmid DNA. Then, I placed the tubes back tubes in a container of crushed ice for 10 minutes.

While I was waiting for the tubes to cool, I labeled my agar plates with a letter that indicates the type of bacteria that they will receive.

Plate	Label
LB/amp	+ pGLO
LB/amp/ara	+ pGLO
LB/amp	- pGLO
LB	- pGLO

I placed the foam microtube holder containing the + pGLO and - pGLO tubes to a 42 degree C water bath for exactly 50 seconds then return the tubes to a container of crushed ice. Tubes should stay in the ice for 2 minutes.

After 2 minutes, I removed microtubes holder from the ice and place them to the foam microtube holder. Then, I added 250 ul of LB broth to each of the bacterial cultures (the + pGLO tube and the - pGLO) with a micropipette and tubes were incubated at room temperature for 10 minutes.

I tapped the closed tubes with finger to mix. Then, I used using a micropipette to transfer 100 ul of the solution from the + pGLO to the surface of the agar in the plates which were labeled +pGLO. A different pipette tips were used to transfer 100 ul of solution from the - pGLO to the surface of the agar in the plates labeled -pGLO. 

I used different sterile loops to spread the mixture over the entire surface of the each agar by quickly skating it back and forth across the plate surface. Plates were stacked and tapped together. I placed the stacked upside down in 37º C incubator for 24 hours.

I checked my plates after 24 hours with UV light and just E. coli witch was cultured on LB/amp/ara plate has been transformed.