Calcium chloride neutralizes
the plasma membrane so DNA
can enter the cell
Place tubes on ice
Ice slows
down the
movement
of the
plasma
membrane
Pick up a colony of bacteria from the
starter plate and submerge it in the CaCl2
until its dispersed (For both tubes)
E. coli is the bacteria being used in this
experiment. It's ideal for transformation because
its small, single-celled, and reproduces quickly
Use a UV light to examine the
pGLO plasmid DNA solution
Immerse an inoculation
loop in the solution and
withdraw a lapful of
plasmid solution
Mix the solution into the +pGLO tube
(ONLY) and return to rack
Incubate the tubes on ice for
10 minutes
While the tubes are
incubating label the four agar
plates as follows:
Heat Shock. Transfer both the
+pGLO and -pGLO into the water
bath, set at 42 C for 50 sec.
Place tubes back on ice and
incubate for 2 minutes
Remove tubes from ice and
add LB broth to both
LB is nutrient broth
(food for bacteria)
Incubate at room
temperature for 10
minutes
Tap the lid of the tube to mix
Pipet transformation and control suspensions to the
corresponding plate
Using a new sterile loop for each plate, spread the suspension evenly
Stack up the plates, tape them together, and put them in the incubator
Heat Shock increases
the bacterial uptake of
foreign DNA
By using the calcium rich environment of the
calcium chloride the heat shock counteracts
the magnetic field between the plasmid DNA
and bacterial cellular membrane thus creating
pores to allow the plasmid DNA to enter the
bacterial cell membrane
Both -pGLO plates serve as control
Goal: Facilitate the replication
of GFP by the pGLO plasmid
The pGLO plasmid consists of not only
for the GFP gene but also the ore, arak
gene, and beta-lactanase
enzyme
This is the enzyme for
ampicillin resistance
Only transformed bacteria will be
able to grow AND glow on the plates
because they contain the genes for
ampicillin resistance and GFP
Plates without amp (only LB
agar will grow but not glow
Ampicillin is a antibiotic
Ori Origin of replication
Allows plasmid replication
araC gene regulates
GFP transcription
through the breakdown
of arabinose
Arabinose is a sugar that
activates the arak gene
through its decomposition
therefore also activating
GFP