GFP Laboratory

(Draw the procedure as you wait for steps )

1. Take a sterile inoculating loop to gently scrape colonies of E.coli

from the starter plate to transfer to your +plasmid and CaCl2 tube.
** Try not to transfer any agar from the plate as well!!**

2. Dip cells on the loop in the tube (the + plasmid and CaCl2 tube) and
SPIN the loop in the solution to dislodge the cell mass. MAKE SURE
THAT THE CELLS ARE NOT STILL STUCK TO THE LOOP!

3. Immediately suspend the cells in the tube by repeatedly pipetting in

and out with a NEW, sterile, pipet.
Look at the tube to make sure that no visible clumps of cells are in the
tube OR stuck in the pipet.
*** The suspension should be CLOUDY!***

4. RETURN THIS TUBE TO ICE!

5. Repeat steps 1-4 with the other tube- the no-plasmid CaCl2 tube.

6. Incubate the tubes on ice for 15 minutes. STOP and draw what you
have done in the spaces for steps one and three.
7. When you’re done drawing, predict what is going to happen for
growth of bacteria to each of the four plates that you are making.
Draw a picture if it helps.
CIRCLE the control plates, put a star next to the experimental.
They are as follows:
a) Luria Broth (LB)/Ampicillin (Amp) +plasmid:
The bacteria in this plate will….

b) LB/Amp - plasmid:

c) LB +plasmid:

d) LB –plasmid:

8. After 15 minutes, “Heat Shock” the cell tubes. Make sure that they
are labeled with your group! Immediately immerse in 42 C water bath
for 90 seconds.

9. Once you have heat shocked the cells, return the tubes to ice for
one or more minutes.
10. Next, use a sterile pipet to add 250 µL Luria Broth (LB) to each
tube. GENLTY tap the tubes with your finger to mix the LB broth with
the cell suspension.

11. Leave the tubes at room temperature for a 5-15 minute recovery
period, depending on how much time is left in class.

12. Now, take up a VERY SMALL portion of your suspension with

another STERILE pipet and add it to the appropriate plate. (+plasmid
to +plasmid plates, - plasmid to –plasmid plates)

13. GENTLY swirl the plates to coat some of the surface of the agar
with your solution.

14. WRAP the edges with parafilm and place at room temperature for
about 36-60 hours at room temperature or incubate at 30 degrees
Celsius for 24-36 hours.
 Data and Analysis for GFP lab

* Write your predictions and (later) write your results from the dishes
on the table below*
Plate: Plate:

Prediction: Prediction:

Results: Results:

Plate: Plate:

Prediction: Prediction:

Results: Results:

1. Observe the colonies through their lids (DO NOT OPEN THE
DISHES!)

2. Count the number of individual colonies and, using a permanent

marker, mark each colony as it is counted. If the cell growth is too
dense to count individual colonies, record, “lawn”. Write any other
interesting things that you see.

LB+plasmid (Positive Control)______________

LB—plasmid (Positive Control)________________

LB/Amp+plasmid (Experimental)________________

LB/Amp—plasmid (Negative Control)_______________

3. What are you selecting for in this experiment? (i.e., what allows you
to identify which bacteria have taken up the plasmid?)

4. What one plate would you first inspect to conclude that the
transformation occurred successfully? Why?

5. Transformation efficiency is the number of antibiotic-resistant

colonies per µg of plasmid DNA. The object is to determine the mass of
plasmid that was spread on the experimental plate and that was,
therefore, responsible for the transformants (number of colonies)
observed.

a. Determine the total mass (in µg) of plasmid used. Remember,

you used 10µL of plasmid at a concentration of 0.005µg/ µL.

Total mass= volume x concentration

b. Calculate the total volume of cell suspension prepared.

c. Now calculate the fraction of the total cell suspension that was
spread on the plate.
 Volume suspension spread/total volume suspension = fraction
spread

d. Determine the mass of plasmid in the cell suspension spread.

Total mass plasmid (a) x fraction spread (c) = mass plasmid DNA
spread

e. Determine the number of colonies per µg plasmid DNA.