Briefing on

GENETIC TRANSFORMATION
using pGLO
Learning Objectives
At the end of the session, the students should be able to:
• Perform correctly the procedure known as
genetic transformation using pGLO plasmid DNA
• Evaluate the effect of pGLO plasmid on E. coli
• Justify the observed changes due to
transformation
• Explain how the genetically transformed bacteria
turned green
• Compute for transformation efficiency
• Discuss importance of gene transformation
Significance of the Experiment
• Genetic transformation is used in many areas
of biotechnology.
• In medicine, diseases caused by defective
genes are beginning to be treated by gene
therapy; that is, by genetically transforming a
sick person’s cells with healthy copies of the
defective gene that causes their disease.
 plasmid

•small DNA molecule within a cell that is physically

separated from a chromosomal DNA
•can replicate independently
•found in bacteria as small, circular, double-stranded DNA
molecules
pGLO plasmid DNA
General Laboratory Rules
• Use sterile technique
Wear sterile gloves and face mask and
protective eyewear / UV rated safety glasses
or goggles
The round circle at the end of the loop, the
tip of the pipet, and the surface of the agar
plate should not be touched or placed onto
fingertips or benchtops or any contaminating
surface.
Eating, drinking, smoking, and applying
cosmetics are not permitted in the work area.
General Laboratory Rules
• Working with E. coli
host organism in the kit is a nonpathogenic
E. coli K-12 strain, the vector containing the
recombinant GFP protein
• Wash hands
after handling materials involving
organisms containing recombinant DNA
molecules
before exiting the laboratory
Methodology: Pre-Lab
• 1. Prepare agar plates (3days prior)

starter plate

LB LB / amp LB / amp LB /amp/arabinose

Methodology: Pre-Lab
• 2. Rehydrate E.coli (1day prior)
Streak starter plate (LB plate)
Rehydrate pGLO plasmid DNA
Methodology: Pre-Lab
• 2. Rehydrate E.coli (1day prior)
Streak starter plate (LB plate)
Rehydrate pGLO plasmid DNA
Methodology: Pre-Lab
• 2. Rehydrate E.coli (1day prior)
Streak starter plate (LB plate)
Rehydrate pGLO plasmid DNA
Methodology: Pre-Lab
• 3. Prepare LB broth aliquot solution (1 day prior)

1ml transformation sol +

1 ml LB broth
 Results of Pre lab

Starter Plate
Methodology: Transformation Lab
1. Label one closed micro TT +pGLO and another
–pGLO. Label both TT with your group number.
Place them in the TT rack
Methodology: Transformation Lab
2. Using a sterile pipet, transfer 250ul of transformation
solution (CaCl2) into each TT.
Methodology: Transformation Lab
3. Place the TT on crushed ice for 15mins. Do not use
cube ice.
Methodology: Transformation Lab
4. Use a sterile loop to pick up a single colony of bacteria
from your starter plate. Immerse the loop into the
transformation solution at the bottom of the +pGLO TT.
Spin the loop between your index finger and thumb until
the entire colony is dispersed in the solution. Place the
TT back in the rack with ice. Using a new sterile loop,
repeat for the –pGLO TT
Methodology: Transformation Lab
5. Immerse a new sterile loop into the pGLO plasmid
DNA. Withdraw a loopful. Mix the loopful into the cell
suspension of the +pGLO TT and mix.

**Shine the UV light onto the original pGLO plasmid DNA,

describe your observation.
Methodology: Transformation Lab
6. Incubate both TT on ice for 10mins. Make sure to
push the TT all the way making contact with the ice.
Methodology: Transformation Lab
7. While the TT are sitting on ice, label your 4 agar plates
on the bottom (not the lid), as shown below
Methodology: Transformation Lab
8. Heat shock. Transfer both the TT into the water bath
set as 42oC, for exactly 50 seconds. Make sure to push
the TT all way down making contact with warm water.
After 50sec, place the TT back on ice immediately.
Incubate TT on ice for 2 mins.
Methodology: Transformation Lab
9. Remove the TT from ice. Using a new sterile pipet,
add 250ul of LB broth to both TT and reclose it.
Incubate the TT at room temperature for 10mins
Methodology: Transformation Lab
10. Gently flick the closed tubes with your finger to mix.
Using a new sterile pipet for each tube, pipet 100ul from
each tube to the corresponding plates
Methodology: Transformation Lab
11. Use a new sterile loop for each plate. Spread the
suspensions evenly around the surface of the agar by
quickly skating the flat surface of a new sterile loop back
and forth across the plate surface.
Methodology: Transformation Lab
12. Stack up your plates and tape them together. Put
your group number and section on the bottom of the
stack. Place the stack upside down in the 37oC
incubator until the next day (18 to 24hrs)
Methodology: Data Collection and
Analysis
Compute for Transformation Efficiency
Quantitative value that describes how effective
you were at getting a plasmid into bacteria
Number of transformed colonies produced per
microgram of DNA added

1. Determine the total number of green

fluorescent cells.
= number of the colonies on your LB/amp/ara
plate
 Compute for Transformation Efficiency
2. Determine the pGLO plasmid DNA on
LB/amp/ara plate
= (total amount of DNA)(fraction of DNA)

Total amount of DNA (ug) Fraction of DNA

= (conc of DNA) (vol of DNA) =volume spread on plate /
= (0.08 ug/ul) (10ul) total volume
= 0.8ug =100ul / 510ul
=0.2
Compute for Transformation Efficiency
3. Compute for transformation efficiency
 Plenary
• The class will discuss the plenary regarding the
experiment
• In the plenary,
Introduction
Learning Objectives
Results
Answers to the guide questions
Summary