LAB 8:
Transgenic Plants II
I. Objectives:
This lab is a continuation of Lab 7. In Part A of today's lab, you will practice running an
agarose gel and will find out if the “test foods” from last time contained transgenes. In Part B
of today’s lab, you will observe the results of the staining to detect reporter gene expression
and draw conclusions about the pattern of expression conferred by the promoter used in these
constructs. By the end of today's lab, students should:

Feel very comfortable with setting up, loading, and running an agarose gel

Understand how GUS reporter gene constructs can be used to test promoter
function in transgenic plants.
II. Safety Considerations

As always - wear nitrile gloves during and after staining the gel to protect yourself
from exposure to ethidium bromide.
III.
Things to Do
PART A. AGAROSE GEL ELECTROPHORESIS OF PCR PRODUCTS
1.
Pour an agarose gel, using the same technique as in Labs 6 and 7. Use 40 mL of
melted agarose. This is a 1.5% gel.
2.
While you are waiting for the gel to cool, retrieve your PCR reactions from Lab 8. Add
8 µl of 6X loading dye to each tube and mix by flicking. We are just loading a portion
of this on the gel, so a few drops on the side will not be a problem
3.
Load your gel with 10 µl of DNA size standards in lanes 1 and 8. Load 15 µl of each
PCR reaction into each lane. Be sure to record the lane number for each sample!
4.
When your samples are loaded, add the gel rig lid and connect the leads to the power
supply. Turn on the voltage to 100 V. Look for the appearance of bubbles on each end
of the rig to confirm that you have a current.
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5.
While you are waiting for your gel to run, continue on to Part B. Keep an eye
on your gel, though, and watch as the dyes in the loading dye begin to separate as
they move through the gel at different rates.
6.
The gel should run for about 45 minutes or until the first dye band has migrated about
2/3 of the way down the gel. Turn off the power supply, disconnect the leads, remove
the lid off the apparatus, and prepare for viewing your gel on the transilluminator.
Remember to hold the gel on either end so that it won't slip off the casting tray as you
carry it to the transilluminator.
7.
Be sure to put on nitrile gloves. Then, stain the gel for 10 min in ethidium bromide as
we did in previous labs.
8.
Carefully pour the used ethidium bromide back into the stock bottle for reuse.
9.
Destain the gel for 10 minutes by submerging it in tap water.
10.
After 10 min. pour the destain into the waste container in the sink (not the ethidium
stock bottle)
11.
Record the results with the gel doc system and make a copy for each lab member’s
notebook.
12.
Dispose of the gel and any contaminated gloves in the waste container immediately
below the gel doc system. Be sure not to touch any clean surfaces, handles, pipets,
yourself, or anything else with potentially contaminated gloves!
13.
Immediately tape your gel image in your notebook and carefully label it with lane
numbers and add a key indicating what is in each lane.
14.
Plot a standard curve using the size standards as we did in Lab 6.
15.
Determine the sizes of the products in each lane and record these.
16.
In your lab notebook, describe what the presence or absence of one or more bands
indicates and why you conclude this. Complete this for each sample lane.
17.
We will discuss this as a group before leaving lab today.
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18.
If you have not already done so, complete Parts B of today's lab.
.
PART B. ANALYSIS OF REPORTER GENE EXPRESSION
1.
Retrieve your tubes of stained plants from the front bench.
2.
Carefully pipette out the GUS staining solution. (The solution is not toxic, but feel free
to wear golves.)
3.
4.
Gently rinse the plants three times with 0.05 M phosphate buffer (pH 7.0)
a.
pipette one mL of buffer per rinse,
b.
gently swirl to rinse
c.
pipette off rinse
d.
leave the plants in the buffer used for the final rinse
Gently transfer 3-4 plants to microscope slides for observation. Place one or two
plants on each slide.
5.
Pipette on approx. 100 µL of glycerol mounting solution and gently cover with a cover
slip (try to avoid bubbles).
6.
Observe the pattern of expression using a compound microscope.
7.
Carefully record the pattern of expression that you observe. Be sure to observe at
higher magnifications also. Record this and your answers to the questions below in
your lab notebook.
8.
In what regions of the plant is the gene expressed?
9.
Can you identify specific tissues and/or cell types in which expression is observable?
Describe these observations in your lab notebook. See Figures 1 and 2 (below) for
information about tissues of the root.
10.
What does this pattern of expression tell us about the activity of the promoter that
was fused to the reporter gene?
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11.
It is proposed that the plant hormome auxin controls the pump in order to regulate
plant growth and development. Does the pattern of expression of the pump here
support this hypothesis?
12.
How might these reporter gene expressing plants be used to further test the
hypothesis that the regulation of this pump by the hormone auxin contributes to the
control of plant growth and development?
V. Lab Clean-Up

Leftover staining and PCR reaction reagents/tubes and tips used for loading the gel
can be discarded in the trash.

All stained agarose gels should be discarded in the ethidium bromide waste container
along with any contaminated gloves.
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Anatomy of Arabidopsis and Onion Root Tips:
The architecture of the root of this model plant is relatively simple (Fig. 1 below). Each of the tissues outside the vascular
bundle (the pericycle, the endodermis, the cortex and the epidermis) are only one cell layer thick.
The columella is one of the gravity sensing organs of the plant. C1-C4 in Figure 1 below refer to specific cells in the
columella.
The QC is the quiescent center. Quiescent means quiet, referring to the fact that these cells seldom divide. The cells next to
the QC do divide frequently and are the stem cells of the root. The cells above the QC give rise to the epidermis, cortex,
endodermis, pericycle and the vascular bundle. The cells below the QC give rise to the root cap, including the columella.
The onion root tip (Figure 2) is comprised of tissues that are more than one cell layer thick. Note the zones of growth and
development indicated on this figure and compare these with the stained areas in your plants.
(From: http://www.plantsci.cam.ac.uk/Haseloff/)
Figure 1. Tissues of the Arabidopsis root
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Figure 2. Primary growth of a root (Fig.
35.13 in Campbell, 8th ed.). Note the location of GUS
expression in the stained plants you are observing in today’s
lab. Does GUS expression correspond with the tissues in
Figure 1 or the zones in Figure 2
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