Background
Transformation is a process by which
bacterial cells take-up or absorb plasmid DNA, causing a change in their
phenotype. E. Coli is commonly used in this process, as the
organism is readily available and easy to work with. To begin, the cells
are first induced, making it easier for them to take-up more DNA than usual, and
therefore the desired plasmid. This is accomplished by adding CaCl2
to the cells and heat shocking them. While the exact process on a
molecular level is not known, it is thought that the calcium ions neutralize the
outer cell membrane and the phosphate ions within the DNA, allowing easier
absorption. Another theory is that because the cell membrane is permeable
to chloride ions but not calcium ions, the chloride ions flood into the
cell accompanied by water molecules. The large amount of water causes the
cell to swell, stretching the cell membrane and allowing DNA uptake to occur
easily. As for heat shock, when subjected to a hot water environment "heat
sensitive" genes are expressed that allow for more efficient DNA uptake.
Protocol
Please see pGLO Transformation
Flow Chart for a simple overview
1) Label 2 microcentrifuge tubes, one -pGLO and the
other +pGLO.
2) Pipette approximately 250μL of CaCl2 into each tube and place them
on ice for 2 minutes.
3) Following icing, obtain an E. Coli colony from the starter plate for
each tube, and mix it into the CaCl2 solution.
4) Withdraw a loop of pGLO plasmid solution and mix into the +pGLO tube ONLY.
Put nothing into the -pGLO tube.
5) Place both tubes on ice to incubate for 10 minutes.
6) While tubes are on ice, label 4 plates
-LB/amp +pGLO
-LB/amp/arab +pGLO
-LB/amp -pGLO
-LB -pGLO
*Note on "amp" and "arab"
7) After 10 minutes, IMMEDIATELY place the tubes in a 42o
water bath for exactly 50 seconds, and then IMMEDIATELY place the
tubes back on ice for 2 minutes.
*This step is crucial to the transformation process,
efficiency is a MUST.
8) Add 250μL of LB broth to both tubes, mix, and incubate at room
temperature for 10 minutes.
*Note on LB broth
9) After 10 minutes, pipette 120μL of the +pGLO suspension onto
each plate labeled +pGLO and 120μL of the -pGLO suspension onto each plate
labeled -pGLO. Spread evenly, and then cover and seal each plate
10) Incubate at 37oC for 24 hours, then observe.