1. Transformation of Bacteria
Transformation occurs when bacteria incorporate foreign DNA into their cell from the environment. Since bacteria is surrounded by a phospholipid bilayer the cell must become competent to receive DNA
Electroporation is a process of inducing the cell to uptake DNA
CaCl2 (calcium chloride) is commonly used as a transforming agent in order to make the cell “chemically competent”.

2. Transformation Procedure
E. coli is harvested in a logarithmic growth phase from an agar plate
E. coli are then exposed to CaCl2 and stressed in ice water
Increases competency
Plasmids are added to the mix
Containing Ampr or bla & a marker such as GFP
Mix is incubated on ice for 15min then transferred to 42oC bath for 2 min to heat shock the bacteria
Increases efficiency of uptake
LB broth is added to suspended colonies and allowed to equilibrate for 10 min
New recombinant cells are plated on fresh plates and incubated at 37oC overnight
On Amp+ and Amp- plates

3. Other methods of DNA uptake
Eukaryotic cells
Transfection using lipids
Plasmids sealed in tiny lipid vesicles are fused with the plasma cell membrane where they release DNA into the cell
Shuttle plasmids are plasmids engineered to infect eukaryotic cells.
A selectable marker (antibiotic resistance gene) such as neomycin and a promotor from a mammalian virus to aid in DNA insertion
CMV (cytomegalovirus) is a human virus that effectively infects many different types of cells

4. Other methods of DNA uptake
Biolistics
Using a DNA coated gold or tungsten fragment to deliver DNA through the cell wall of a plant

5. Selection of Transformation
Color
GFP or GRP are fluorescent markers
pUC18 contains the lacZ gene that grows blue colonies when grown on galactose medium
An MCS site is incorporated into the lac Z gene to knock out the lacZ gene when DNA is successfully inserted making the colonies white
Antibiotic resistance
Added into cloning vectors
Lethal genes
pJET1.2 includes a gene for a lethal protein the kills E. coli
An MCS site is inserted within the gene so that it is turned off when DNA is successfully inserted
Selection of Transformation

6. Transformation efficiency
Transformation efficiency is measured colony forming units per micrograms of plasmid DNA (CFU/mg)
Efficiency is affected by all variables
Bacteria used, Electroporation method, Heat shock temp and timing, Plasmid amount, size, and type, …
Calculating efficiency… 10ng is transformed into 500ml. 10ml of that solution is then spread onto an agar plate and incubated and transformed cells counted.
Calculation
DNA spread (mg) = (volume spread(ml) x DNA wt(mg)
total transformation volume (ml)
10(ml) x 0.05(mg) = 0.001(mg)
500(ml)
CFU/mg is # of colonies/DNA spread
196/0.001(mg) = 1.96 x 105 CFU/mg
Transformation efficiency