MBIO

3812

Ex 10-3 Bacterial Transformation: the pGLO System

Prepartion of Competent E. coli cells
1. Label 2 microtubes, one as + plasmid and the other as no plasmid.

2. Add 250 l of CaCl2 (calcium chloride) to each tube and place the tubes in a tray of ice.

3. Using a sterile plastic loop remove several colonies from a plate of E. coli and transfer
these bacteria to the microtube marked as + plasmid making sure the bacteria are
resuspended well.

4. Using a new sterile plastic loop remove several more colonies from a plate of E. coli
and transfer these bacteria to the microtube marked as no plasmid making sure the
bacteria are resuspended well.

5. Return both microtubes to the tray of ice


Addition of Plasmid DNA and Heat Shocking
1. Using a new sterile 10 l volumetric plastic loop take a loopful of the pGLO plasmid
from the tube at the front bench of the lab and add it to the microtube labeled +
plasmid. Do not add anything to the microtube labeled no plasmid.

2. Incubate both microtubes on ice for 10 minutes.

3. Once the 10 minutes is up move both tubes to the 42C H2O bath at the back of the lab
for 1 minute.

4. After the minute is up place both tubes back in the ice tray for 2 minutes.

5. After the 2 minutes is up move both tubes to room temperature by placing them in a
rack on the lab bench.


Bacterial Recovery and Plating
1. Add 250 l of LB broth to both microtubes.

2. Incubate the tubes at room temperature on the bench for 10 minutes.

3. Using a glass hockey stick make the following spread plates:
(i) LB plate 100 l from the no plasmid tube
(ii) LB/ampicillin plate 100 l from the no plasmid tube
(iii) LB/ampicillin plate 100 l from the + plasmid tube
(iv) LB/ampicillin/arabinose 100 l from the + plasmid tube