4222314
Description
Mind Map by Tashelle Davis, updated more than 1 year ago
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Created by Tashelle Davis
over 8 years ago
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Bacterial Transformation
- Label two micro test tubes
- +pGLO and -pGLO
- Add transformation solution (CaCl2)
- 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
- Stack up the plates, tape them together, and put them in the incubator
- Using a new sterile loop for each plate, spread the suspension evenly
- Pipet transformation and control suspensions to the
corresponding plate
- Tap the lid of the tube to mix
- LB is nutrient broth
(food for bacteria)
- Remove tubes from ice and
add LB broth to both
- 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
- 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
- Place tubes back on ice and
incubate for 2 minutes
- Heat Shock. Transfer both the
+pGLO and -pGLO into the water
bath, set at 42 C for 50 sec.
- While the tubes are
incubating label the four agar
plates as follows:
- Incubate the tubes on ice for
10 minutes
- Mix the solution into the +pGLO tube
(ONLY) and return to rack
- Immerse an inoculation
loop in the solution and
withdraw a lapful of
plasmid solution
- E. coli is the bacteria being used in this
experiment. It's ideal for transformation because
its small, single-celled, and reproduces quickly
- Ice slows
down the
movement
of the
plasma
membrane
- Calcium chloride neutralizes
the plasma membrane so DNA
can enter the cell
- +pGLO and -pGLO
- 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
- Plates without amp (only LB
agar will grow but not glow
- Ampicillin is a antibiotic
- Only transformed bacteria will be
able to grow AND glow on the plates
because they contain the genes for
ampicillin resistance and GFP
- Ori Origin of replication
- Allows plasmid 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
- Arabinose is a sugar that
activates the arak gene
through its decomposition
therefore also activating
GFP
- This is the enzyme for
ampicillin resistance
- The pGLO plasmid consists of not only
for the GFP gene but also the ore, arak
gene, and beta-lactanase
enzyme
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