Lab: Bacterial Transformation with pGreen
or transgenic science
is a process that molecular biologists use to
insert a foreign piece of DNA into a host cell such as bacteria, yeast, animal or plant cells. A
biotechnology company formerly known as
was the first to make human insulin from a
The gene for human insulin was isolated
special circular piece of DNA called a plasmid
The plasmid is then inserted into the host bacterium,
by a technique called “heat shocking”.
The bacteria recognize and
the genes on th
Every time the cell divides (20 minutes) the offspring inherited the plasmid, making
This is how many human hormones, such as
insulin and human growth hormone (HGH
transformation lab that you will perform involves a plasmid called pGreen. It has two
genes on it. One for resistance to the antibiotic called
This is a type of
let scientists know the bacteria have been transformed. The second gene is for bioluminescence that
will cause the bacteria to glow a green color in the dark. This
too serves as a
is genetically designed
American geneticist Matthew Meselson. It was primarily
developed to be a competent host cell type to accept a foreign piece of DNA. It will not pose any
health risk to any organism. This cell is used worldwide in research institutions, universities, a
To genetically transform the
by inserted the pGreen plasmid.
Groups of 4 students
1 LB only plate
Cup of ice
42O water bath
1 LB/AMP plate
Note: Unit of
measurement for the micropipette is microliters (ul). 1000ul = 1ml
1. Wipe lab table with 10%
solution or detergent wipe before beginning work.
2. Set large micropipette (p1000
or a p200)
and with a sterile tip, transfer 250 ul of CaCl2 to clear
Note, if using p200 set device for 200 ul, and then repeat with setting at 50 ul.
3. Place tube on ice.
4. Go up to demo table and teacher will assist you in using a sterile disposable loop to transfer one or
two large bacterial colony into
5. With a new tip, use the large
suspend the cells by pipetting up and down slowly.
Hold tube up to light to check if cell mass has been re
6. Place the tube back on ice.
7. Using the small micropipette (p2
0) with a sterile tip, add 10 ul of the pGreen DNA plasmid directly
into the cell suspension. Tap the side of the tube with your finger to mix.
8. Place the tube back on ice and incubate for 5 to 10 minutes.
9. While the cells are incubating, use a
permanent marker to label the bottom edge of the LB plate
with your team name, period and LB. Label
the LB/AMP plate (has red stripe on side) the same way
but with LB/AMP. See diagrams below.
Carry the ice cup with cell suspension tube in it over to the 42O water bath.
Immerse the tube in the water bath for
exactly 90 seconds.
11. Immediately return the tube to ice for
at least 1 minute
for the cells to recover. Remove and hold
at room temperature for the rest of the lab.
12. Use a large micropipette (p1000
) with a sterile tip and add 250 ul
of LB broth to the tube.
Gently tap the tube with your finger to mix.
13. With a new tip
, transfer 100 ul of the cell suspension from the tube to the center of the LB plate.
Also transfer 100 ul of the cell suspension from the tube to the center of t
he LB/AMP plate.
14. Gently spread the suspension evenly across the LB plate using a clean sterile disposable loop. Go
back and forth while turning the plate in a clockwise motion.
BE CAREFUL NOT
to penetrate the
surface of the nutrient agar.
. Repeat the spreading process on the LB/AMP plate. You may use the same loop. Bring the loop
to the front of the classroom and insert in Erlenmeyer flash with bleach for disposal.
16. After 2 minutes tape both plates closed. Seal the plates with masking tape and place them
UPSIDE DOWN in the 37OC incubator for 24 hours.
Petri dish with
Label the under side
LB only plate
Has Red stripe
Name____________________________________________Period_____ Due Date________________
Lab: Bacterial Transformation with pGreen
Draw your LB only and LB/AMP plate growth results.
Label plate the transformed bacteria.
1. Which plate is the positive control? ______________________________________________.
2. Which plate is the experiment plate? ______________________________________________.
3. What is different in the experim
ental plate from the control plate? ______________________.
4. On which plate do non
transformed and transformed bacteria grow? ____________________
5. On the plate for question 4, why don’t you see both types of bacteria? ____________________
On which plate do transformed bacteria grow well
why must they be
formed” to survive on this plate?
What is the purpose of using a plasmid
with the bioluminescence gene