Greenomes – Plant Molecular Genetics and Genomics
Detecting Genetically Modified Food by PCR
Most Americans would probably be surprised to learn that more than 60% of fresh vegetables and
processed foods sold in supermarkets today, have been altered by direct gene transfer. Over 150 million
acres of farmland worldwide are used to grow genetically modified (GM) crops. The most widely planted
GM crops are corn, soybeans, and cotton.
This laboratory uses a rapid method to isolate DNA from plant tissue and to genotype a soy plant
using the polymerase chain reaction (PCR) method. The 35S promoter, which drives expression of the
glyphosate resistance gene, is identified in soy plants that are resistant to the herbicide "Roundup”.
Resistance correlates with an insertion allele that is readily separated from the wild-type allele by
electrophoresis on an agarose mini-gel. The technique can then be extended to assay common foods for
molecular evidence of genetic modification.
Objectives/Goals:
This laboratory provides practical insight into:
 the relationship between genes, proteins, and traits.
 three methods (DNA extraction, PCR, and gel electrophoresis) that are commonly used in
biological research.
 gene transfer technology and its impact on society.
INTRODUCTION
Methods in genetic engineering are producing a revolution in agriculture. Genes that encode for
herbicide resistance, insect resistance, draught tolerance, frost tolerance, and other traits have been
added to numerous plants of commercial importance- including strawberries, soy, corn, potatoes, rice,
wheat, and canola.
A common approach to transferring new genes into different plant species makes use of the
pathogenic plant bacterium Agrobacterium tumefaciens. In the wild, infection by this bacterium causes
plant tumors, called crown galls. These tumors provide nutrients for the bacteria’s successful colonization
of the plant. Galls are produced by the expression of genes contained on Agrobacterium’s tumor inducing
(Ti) plasmid. The Ti plasmid contains a region of oncogenic DNA, flanked by direct repeats, that is
transferred to the host cell. This mobile region of the Ti plasmid, called T (transfer) DNA, inserts randomly
into the plant cell genome. Once inserted into the plant genome, the infected cell begins to express genes
located on the T-DNA. The host cell then expresses genes on the T DNA needed by the pathogen. By
placing DNA of interest within a modified T DNA region, researchers are able to use Agrobacterium to
introduce foreign DNA into plants.
Agrobacterium-mediated gene transfer involves the use of two plasmids and is referred to as a
“binary system”. First, the gene of interest is cloned into a small plasmid. This plasmid contains the direct
repeat regions of the Ti plasmid, which allows efficient transfer of DNA into the plant cell genome. A
polylinker containing multiple recognition sites allows one to splice a gene of interest between the direct
repeats. The gene is inserted adjacent to a strong promoter-such as the 35S promoter from cauliflower
mosaic virus. The plasmid also contains a selectable marker, the kanamycin-resistance gene, which
allows successfully transformed plants to be identified by antibiotic selection. After subcloning the gene of
interest, the plasmid is purified from E. coli and inserted into a strain of Agrobacterium that contains the
second plasmid of the binary system. This plasmid is a larger, modified form of Ti, which carries genes
required for infection and T-DNA transfer.
The genetically modified Agrobacterium is then used to infect a host plant. In Arabidopsis, ovules
can be transformed simply by dipping early stage flowers in a suspension of Agrobacterium. After
fertilization, a small proportion of seeds will produce genetically modified offspring.
Agriculture has been greatly impacted by the advances in gene transfer methods. One example
has been the introduction of a gene that provides resistance to the broad-spectrum herbicide glyphosate,
commonly known as “Roundup”. Crops genetically altered to possess this gene are referred to as
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“Roundup Ready”. These crops are resistant to the herbicide, while the invasive weeds are not. The
major advantages of the "Roundup Ready” system include better weed control, reduction of crop injury,
higher yield, and lower environmental impact than traditional herbicide systems.
REFERENCES
Castle, L.A., Siehl, D.L., Gorton, R., Patten, P.A., Chen, Y.H., Bertain, S., Cho, H.J., Wong, N.D., Liu, D.,
Lassner, M.W. (2004). Discovery and Directed Evolution of a Glyphosate Tolerance Gene. Science
304: 1151-1154.
Edwards, K., Johnstone, C. and Thompson, C. (1991). A Simple and Rapid Method for the Preparation of
Plant Genomic DNA for PCR Analysis. Nucleic Acids. Res.19: 1349.
Stalker, D.M., McBride, K.E., Maiyj, L.D. (1988). Herbicide Resistance in Transgenic Plants Expressing a
Bacterial Detoxification Gene. Science 242: 419-423.
Vollenhofer, S., Burg, K., Schmidt, J., Kroath, H. (1999). Genetically Modified Organisms in Food
Screening and Specific Detection by Polymerase Chain Reaction. J. Agric. Food Chem. 47: 50385043
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REAGENTS, SUPPLIES, AND EQUIPMENT
Reagents
Wild-type and Roundup Ready soybean seed
Assorted dry food products
Edward’s buffer, 4 mL
Isopropanol, 1.5 mL
Tris/EDTA (TE) buffer, 400 L
35S1/35S2 primer/loading dye mix*, 75 L
Tub5/Tub3 primer/loading dye mix*, 75 L
Ready-To-GoTM PCR beads
Mineral oil, 5 mL (depending on thermal
cycler)
Marker, pBR322/BstN1* (9.75 g), 130L
10X TBE, 300 mL
Agarose, 2 g
Ethidium bromide (1 g/mL), 250 mL
or
CarolinaBLU gel and buffer stain, 7 mL
CarolinaBLU final stain, 250 mL
*Store in a -20C freezer
Supplies and Equipment
Seed growing tray
Planting container
Potting soil
Pellet pestles
Permanent markers
1.5 mL microcentrifuge tubes
Micropipets and tips (for measuring volumes from 2.5 L
to 900 L)
Microcentrifuge tube racks (or empty pipet tip boxes)
Microcentrifuge
Vortex (optional)
Ice buckets with crushed ice
Thermal cycler
Water bath or heating block for boiling samples
Gel electrophoresis chamber and power supply
Staining trays
Latex gloves
White light box (to visualize DNA with CarolinaBLU)
UV transilluminator (to visualize DNA with ethidium
bromide)
LAB FLOW AND SUMMARY
Note: You must plant the soy seed 2-3 weeks prior to performinging the lab.
This lab can be broken into three parts:
I. Isolating DNA from soy plant tissue and dry food products using Edward's buffer.
II. Amplifying the 35S promoter and tubulin locus by PCR. During this step, specific primers are used to
analyze the wild-type and the transformed plants having the 35S promoter.
III. Analyzing the amplified DNA by agarose gel electrophoresis.
The following table will help you to plan and integrate the three parts of the experiment.
Part
Day
Time
Preparation of
soy plants
2-3
weeks
before
lab
15-30 min.
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Activity
Plant soy seeds
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I. DNA Isolation
1
II. PCR Amplification
2
III. Analyzing Amplified
DNA by Gel
Electrophoresis
3
4
30 min.
30-60 min.
30-60 min.
15-30 min.
70+ min.
30 min.
30 min
30+ min.
20+ min.
20 min. to
overnight
20 min.
Pre-lab: Set up student stations
Isolate soy DNA
Pre-lab: Set up student stations
Set up PCR reactions
Post-lab: Amplify DNA in thermal cycler
Prepare agarose gel solution and cast gels
Load DNA samples into gels
Electrophorese samples
Post-lab: Stain gels
Post-lab: De-stain gels
Post-lab: Photograph gels
GROWING SOY
The table below provides a list of items for growing soy plants. Most of the supplies are from Hummert but
many of them can be substituted with products that are easily obtainable from a hardware store.
Item
Shelving
Fluorescent light fixtures
and bulbs
Timer
Soil
Trays
Pots
Description
Any shelves can be used that can house
fluorescent light fixtures so that lights are 12-18
inches from the plants.
Light fixtures should hold at least two 40 watt
fluorescent bulbs. The lights should be
“daylight” lights as opposed to cool white lights.
A timer is needed if plants are to be maintained
on a cycle of 16 hours of light and 8 hours of
dark. Plants can also be grown in 24 hours of
light if a timer is not available.
The potting soil to grow soy is Metro Mix 200
(Catalog # 10-0325). Fertilizer and insecticide
can be added as required.
Soy plants are watered from above so planting
pots should be placed in plastic tray with holes
(Catalog # 11-3000-1). The industry standard
for trays is a 10” X 20” plastic flat. The planting
pot inserts fit directly into these trays.
The inserts for the trays commonly used
contain 8 planting pots (Catalog # 11-0250-1).
There are also other inserts that can be used if
a different number of planting pots are required.
Supplier
Hardware store
Hardware store
Hardware store
www.hummert.com
www.hummert.com
www.hummert.com
Planting Seeds
Obtain wild-type and Roundup Ready soy seed. Plant seeds as described below and allow for a 2-week
growth period.
1. Place the planting pots into a plastic tray containing holes. The holes will allow excess water to drain
from the soil when it is initially dampened. Fill the planting pots evenly with soil but do not pack the soil
tightly. Note: For the best results, obtain or make a potting soil formulated specifically for growing soy
seed.
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2. Plant the seeds 0.5 inches below the soil using your finger. Leave 3 to 4 inches of space between
them to allow optimal growth and to easily visualize the plant phenotype.
3. Place the planting container into the growing tray. Water the plants from above to prevent the soil
from drying out. However, do not allow the soil to remain soggy.
4. Grow the plants close to a sunny window at room temperature or slightly warmer.
5. Harvest plant tissue for PCR as soon as the plant’s first leaves become visible. This should be about 2
weeks after planting – depending on light and temperature conditions.
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PART I: ISOLATING DNA FROM SOY AND DRY FOOD PRODUCTS
Pre-lab Notes
Dry Food Products
Dry food products work best using the DNA extraction protocol outlined below. Food products should
contain either soy or corn as an ingredient. Products that have been tested successfully using this
procedure include Doritos brand tortilla chips, Tostitos blue corn chips, Betty Crocker Bacos, Jiffy corn
muffin mix, Pepperidge Farm Sausalito cookies, Almased multi-protein powder, and Meow Mix cat food.
Pre-lab Set Up
Each station serving two students working as a team should have:
Wild-type and Roundup Ready soy plants
Dry food product
Edward's buffer, 4 mL
Isopropanol, 1.5 mL
Tris/EDTA (TE) buffer, 400 L
6- 1.5 mL microcentrifuge tubes
Permanent marker
3- Disposable pellet pestles
20-200 L micropipet and tips
100-1,000 L micropipet and tips
Microcentrifuge
Water bath or heating block to boil samples
Vortex (optional)
Ice bucket with crushed ice
Procedure
1. Obtain one wild-type and one Roundup Ready soy plant. From each plant, take two pieces of leaf
tissue approximately 1/4 inch in diameter. (The large end of a 1,000 L pipet tip will punch disks of
this size.) Place the leaf tissue in separate microcentrifuge tubes, and label with plant type and your
group number.
2. For dry food products, obtain a 2-3 mm piece of food product and place in a separate microcentrifuge
tube, and label with sample type and group number.
3. Add 100L of Edward’s buffer to each tube containing the plant or food material. Grind the plant tissue
or food product forcefully in the microcentrifuge tubes using separate pellet pestles. Grind for
approximately 1 minute. The plant tissue sample should become green when it is fully ground.
4. Add 900 L of Edward's buffer to each tube containing the ground sample. Grind briefly to remove
tissue from the pellet pestle and to liquify any remaining pieces of sample.
5. Vortex the tubes for 5 seconds, by hand or machine. Boil the samples for 5 minutes in a water bath or
heating block. Note: Be sure to monitor the samples as the eppendorf tube lids may open as the
tubes heat.
6. Microcentrifuge the tubes containing the ground plant tissue or food sample for 2 minutes. After 2
minutes any insoluble material should form a tight pellet at the bottom of the tubes.
7. Transfer 350 L of each supernatant to a fresh tube. The supernatant contains the desired DNA;
make sure not to disturb the pelleted material when transferring the supernatant. (This is best
accomplished by pipetting several times using a medium micropipet set at 100 L.)
8. Add 400 L of isopropanol to each of the DNA-containing supernatants. Mix, and leave at room
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temperature for 3 minutes. This step precipitates the DNA.
9. Microcentrifuge the tubes with the isopropanol and supernatant for 5 minutes with the hinge of tubes
pointing outward.
10. After centrifugation, the pellet should be located at the bottom-side of the tubes underneath the hinge.
It may be teardrop shaped or appear as small, scattered granules. Don’t be concerned if you can’t see
a pellet. Carefully pour off the supernatant, then completely remove the remaining liquid with a
medium pipet set at 100 L.
11. Air dry the pellets for 10 minutes to remove any remaining isopropanol.
12. After drying, resuspend each DNA pellet in 100 L of TE buffer. Pipet in and out, taking care to wash
down the side of the tube underneath the hinge, where the DNA has accumulated during
centrifugation.
13. Centrifuge the DNA/TE solution for 1 minute to pellet any material that did not go into solution. You will
use 2.5 L of the supernatant as the template DNA for the PCR reactions.
14. DNA may be used immediately or stored at -20C. Keep the DNA on ice during use.
PART II: AMPLIFYING DNA BY PCR
Pre-lab Notes
This procedure uses two PCR reactions to analyze each plant or food product. One primer set
(35S1/35S2) amplifies the 35S promoter of cauliflower mosaic virus. This promoter is used to drive
expression of the glyphosate resistance gene used in Roundup Ready crops. The second set (Tub5/Tub3)
amplifies a fragment of a tubulin gene that is present in all plants. Therefore, the primer set amplifying the
tubulin gene is useful as a positive control for template DNA quality. It helps insure that any lack of
amplification by the first primer set is a result of the absence of the 35S promoter – not a problem of
template quantity or quality. The PCR products from the two primer sets are similar in size: 187 bp for the
Tub5/Tub3 primer set and 162 bp for the 35S1/35S2 primer set. Therefore, these PCR products must be
loaded in separate lanes of a 2% agarose gel during the electrophoresis part of this experiment.
Ready-To-Go PCR BeadsTM
Each PCR bead contains reagents so that when brought to a final volume of 25 L the reaction
contains 2.5 units of Taq polymerase, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, and 200 M
of each dNTP.
Primer/Loading Dye Mix
This mix incorporates the appropriate primer pair (0.25 picomoles/L of each primer), 13.9% sucrose,
and 0.0082% cresol red. The inclusion of loading dye components, sucrose and cresol red, allows the
amplified product to be directly loaded into an agarose gel for electrophoresis.
Setting Up PCR Reactions
The lyophilized Taq polymerase in the Ready-To-Go PCR Bead™ becomes active immediately upon
addition of the primer/loading dye mix. In the absence of thermal cycling, “nonspecific priming” allows the
polymerase to begin generating erroneous products, which can show up as extra bands in gel analysis.
Therefore, work quickly. Be sure the thermal cycler is set and have all experimenters set up their
PCR reactions as a coordinated effort. Add primer/loading dye mix to all reaction tubes, then add
each student template, and begin thermal cycling immediately. Hold reactions on ice until ready
to load into the thermal cycler.
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Greenomes – Plant Molecular Genetics and Genomics
Thermal Cycling
PCR amplification from crude cell extracts is biochemically demanding, and requires the precision of
automated thermal cycling. However, amplification of the 35S and tubulin loci is not complicated by the
presence of repeated units. Therefore, the recommended amplification times and temperatures will work
adequately for all types of thermal cyclers.
To hand amplify, simply set up three constant temperature water baths (or heat blocks) at 94C,
60C, and 72C. Secure the student reactions in a test tube rack, and rotate the rack successively through
the three baths for 30 seconds each. Stop after 32 cycles.
Pre-lab Set up
Each station serving two students working as a team should have:
Wild-type and Roundup Ready soy DNA, from Part I (on ice)
Food Product DNA, from Part I (on ice)
75 L 35S1/35S2 primer/loading dye mix (on ice)
75 L Tub5/Tub3 primer/loading dye mix (on ice)
6 Ready-To-Go PCR Beads™ (in reaction tubes)
Permanent marker
1-20 L micropipet and tips
20-200 L micropipet and tips
Thermal cycler
Mineral oil, 5 mL (depending on thermal cycler)
Ice bucket with crushed ice
Procedure
1. Use a micropipet with a fresh tip to add 22.5 L of the 35S1/35S2 primer/loading dye mix to each of 3
PCR tubes containing a Ready-To-Go PCR Bead™. Tap the tube very gently with your finger to
dissolve the bead. Label each tube with primer set and group number. Then label one tube “WT”
(wild-type), one tube “RR” (Roundup Ready.), and one tube “FP” (food product).
2. Use a micropipet with a fresh tip to add 22.5 L of the Tub5/Tub3 primer/loading dye mix to each of 3
PCR tubes containing a Ready-To-Go PCR Bead™. Tap the tube very gently with your finger to
dissolve the bead. Label each tube with primer set and group number. Then label one tube “WT”
(wild-type), one tube “RR” (Roundup Ready.), and one tube “FP” (food product).
3. Use a micropipet with a fresh tip to add 2.5 L of wild-type soy DNA (from Part I) to each of the two
reaction tubes marked “WT”. Mix by gently pipeting up and down. If necessary, pool the reagents by
pulsing in a microcentrifuge or by sharply tapping the tube bottom on the lab bench.
4. Use a micropipet with a fresh tip to add 2.5 L of Roundup Ready soy DNA (from Part I) to each of the
two reaction tubes marked “RR”. Mix by gently pipeting up and down. If necessary, pool the reagents
by pulsing in a microcentrifuge or by sharply tapping the tube bottom on the lab bench.
5. Use a micropipet with a fresh tip to add 2.5 L of food product DNA (from Part I) to each of the two
reaction tubes marked “FP”. Mix by gently pipeting up and down. If necessary, pool the reagents by
pulsing in a microcentrifuge or by sharply tapping the tube bottom on the lab bench.
6. If necessary, add one drop of mineral oil to the top of the reactants in the PCR tubes. Be careful not to
touch the dropper tip to the tube or reactants, or subsequent reactions will be contaminated with DNA
from your preparation. Note: Thermal cyclers with heated lids do not require the use of mineral oil.
7. Store all samples on ice or in the freezer until you are ready to amplify. Program the thermal cycler for
32 cycles with the following cycle profile. The program may be linked to a 4°C hold program after the
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32 cycles are completed.
Denaturation step:
Annealing step:
Extension step:
94C
60C
72C
30 sec
30 sec
30 sec
8. After cycling, store the amplified DNA at -20°C until you are ready to continue with Part III.
PART III: ANALYZING AMPLIFIED DNA BY GEL ELECTROPHORESIS
Pre-Lab Notes
An objective in this experiment is to let students determine the genotype of the individual plants. The
students can also pool their data and use the segregation ratio of the genotypes to determine the
genotype of the parental plant.
Cresol Red Loading Dye
The cresol red and sucrose in the primer mix function as loading dye, so that amplified samples can
be loaded directly into an agarose gel. This is a nice time saver. However, since it has relatively little sugar
and cresol red, this loading dye is more difficult to use than typical loading dyes. So, encourage students
to load very carefully.
DNA Size Markers
Plasmid pBR322 digested with the restriction endonuclease BstN I is an inexpensive marker and
produces fragments that are useful as size markers in this experiment. The size of the DNA fragments in
the marker are 1,857 bp, 1,058 bp, 929 bp, 383 bp, and 121 bp. Use 20 L of this DNA ladder per gel.
Other markers or a 100 bp ladder may also be substituted.
Viewing and Photographing Gels
View and photograph gels as soon as possible after appropriate destaining. Over time, small-sized
PCR products diffuse through the gel and become unclear. If kept refrigerated in a very small amount of
distilled or deionized water, CarolinaBLU™ stained gels will retain their integrity for months.
Pre-lab Set Up
Depending upon your situation, you may wish to prepare the 1X TBE buffer, the 2% agarose, or the
agarose gels ahead of time for your students. Instructions on how to prepare the 1X TBE buffer, the 2%
agarose, and the agarose gels have been included with the student instructions should you decide to have
your students perform these procedures. The set up below is described as though the instructor prepared
the reagents, but not the gel, ahead of time.
Each station serving two students working as a team should have:
Wild-type soy 35S and tubulin PCR products from Part II (on ice)
Roundup Ready soy 35S and tubulin PCR products from Part II (on ice)
Food product 35S and tubulin PCR products from Part II (on ice)
pBR322/BstNI markers (on ice)
2% agarose in 1X TBE
1X TBE buffer
1 g/mL ethidium bromide or
CarolinaBLU staining solution
2-20 L micropipet and tips
20-200 L micropipet and tips
Electrophoresis chamber and power supply
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Staining tray
Transilluminator with camera
Ice bucket with crushed ice
Procedure
1. Prepare a 1X concentration of TBE by diluting 300 mL of 10X concentrated stock into 2,700 mL of
deionized or distilled water. Mix thoroughly.
2. Prepare a 2% agarose gel in 1X TBE by adding 2 g of agarose to 100 mL of 1X TBE in a 500 mL
flask or beaker. Heat flask in a boiling water bath (approximately 15 minutes) or in a microwave oven
(approximately 4 minutes) until the agarose is completely dissolved. You should no longer see
agarose particles floating in solution when the beaker is swirled. Allow the agarose to cool to 5565C. You should be able to touch the container without burning yourself while pouring it into the gel
tray.
3. Seal the ends of the gel tray with masking tape and insert the comb. Pour cooled agarose solution
into the tray to form a gel approximately one quarter inch thick. Allow the gel to solidify completely.
The gel should appear cloudy when completely solidified. This takes approximately 20 minutes.
4. Place the gel into the electrophoresis chamber, and add enough 1X TBE buffer to cover the surface of
the gel. Carefully remove the comb, and add additional 1X TBE buffer to just cover and fill in wells,
creating a smooth buffer surface. (Do not add more buffer than necessary. Too much buffer above
the gel channels electrical current over the gel, increasing running time.)
5. Use a micropipet with a fresh tip to add 25 L of each of the sample/loading dye mixtures into your
assigned wells of a 2% agarose gel. (If you used mineral oil during PCR, pierce your pipet tip through
the layer of mineral oil to withdraw the PCR products and leave the mineral oil behind in the original
tube.) Important: Expel any air from the tip before loading, and be careful not to push the tip of the
pipet through the bottom of the sample well.
6. Load 20 L of the molecular weight marker (pBR322/BstN1) into one well.
7. Run the gels at 130 V for approximately 30 minutes. Adequate separation will have occurred when the
cresol red dye front has moved at least 50 mm from the wells.
8. Once the loading dye has migrated an appropriate distance through the gel, process the gel by
soaking it in stain. If you are using ethidium bromide, stain for 15 minutes. If you are using
CarolinaBlu, see below. Use gloves when handling ethidium bromide or anything that has
ethidium bromide on it. Ethidium bromide is a known mutagen and care should be taken when
using and disposing of it.
9. View gel using transillumination and photograph.
Staining with CarolinaBLU
To stain gels following electrophoresis, cover the gel with the CarolinaBLU Final stain and let sit for
20-30 minutes. Agitate gently (optional). Pour the stain back into the bottle to be used again. (The stain
can be used 6-8 times.) Cover the gel with deionized or distilled water to destain. Use distilled or deionized
water since the chloride ions present in tap water can partially remove the stain from the DNA bands and
will cause the staining to fade. Change the water 3-4 times over the course of 30-40 minutes. Agitate the
gel occasionally. Bands that are not immediately present will become more apparent with time and will
reach their maximum visibility if the gel is left to stain overnight in just enough stain to cover the gel. Gels
left overnight in a large volume of water may destain too much.
CarolinaBLU can also be used to stain the DNA while the gel is being run. The staining will not be as
intense as the final stain, and final staining will still be required. However, staining while the gel is running
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may slightly increase the sensitivity of the stain and may allow the students to visualize their results prior to
the end of the gel run.
To stain the gel while it is running, add the CarolinaBLU Gel and Buffer Stain in the amounts
indicated below. Note that the amount of stain added is dependent upon the voltage used for
electrophoresis. Do not use more stain than recommended. This may precipitate the DNA in the
wells and create artifact bands. Gels containing CarolinaBLU may be prepared one day ahead of the
lab day if necessary. However, gels stored longer tend to fade and lose their ability to stain DNA bands
during electrophoresis.
Use the table below for the addition of CarolinaBLU gel and buffer stain to agarose solutions.
Voltage
<50 Volts
>50 Volts
Agarose Volume
Stain volume
30 mL
40 L (1 drop)
200 mL
240 L (6 drops)
400 mL
520 L (13 drops)
50 mL
80 L (2 drops)
300 mL
480 L (12 drops)
400 mL
640 L (16 drops)
Use the table below for the addition of CarolinaBLU stain to the 1X TBE buffer.
Voltage
Buffer Volume
Stain volume
500 mL
480 L (12 drops)
3 liters
2.9 mL (72 drops)
500 mL
960 L (24 drops)
<50 Volts
>50 Volts
2.6 liters
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RESULTS AND DISCUSSION
1. Observe the photograph of the stained gel containing your sample and those from other students.
Orient the photograph with wells at the top. Interpret each lane of the gel. Use the sample gel pictured
below to help you.
a. Scan across the photograph of your gel and others as well to get an impression of what you see
in each lane. You should notice that virtually all student lanes contain one or two prominent
bands.
b. Now locate the lane containing the pBR322/BstN I markers on the left hand side of the gel.
Working from the well, locate the bands corresponding to each restriction fragment: 1,857bp,
1,058 bp, 929 bp, 383 bp, and 121 bp (may be faint or not visible at all). Alternatively, locate the
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lane containing the 100 bp ladder on the right hand side of the gel. These DNA markers
increase in size in 100 bp increments starting with the fastest migrating band of 100 bp.
c.
The amplification product of the 35S promoter (162 bp), which has been amplified using the
35S1/35S2 primers, should align between the 121 bp marker and the 383 bp marker of
pBR322/BstNI markers (or between the 100 bp and 200 bp markers of the 100 bp ladder). The
amplification product of the tubulin gene (187 bp), which has been amplified using the Tub5/Tub3
primers, should align between the 121 bp marker and the 383 bp marker (or between the 100 bp
and 200 bp markers of the 100 bp ladder).
d. Establish the exact size of your PCR products as follows.
i.
Measure the distance that each marker DNA band migrated from the sample well by
measuring from the front of each well to the front edge of each band. The sizes of the DNA
fragments in your pBR322/BstNI marker are as follows: 1,857bp, 1,058 bp, 929 bp, 383 bp,
and 121 bp. Should you use another marker, adjust the numbers accordingly.
ii.
Set up semi-log graph paper with the x-axis as the distance migrated by the DNA fragments
and the y-axis (the logarithmic axis) as base pair length of the fragments. Plot the distance
migrated versus base-pair length for each marker DNA fragment and connect the data points
with a line – this line will be referred to as the “standard curve.” There is a linear relationship
between the distance that a DNA band travels and the log of its molecular weight. Since
weight of a DNA fragment is proportional to the number of base pairs in the fragment, base
pairs are frequently used in place of molecular weight when these types of calculations are
done with DNA. Also note that since you are plotting the molecular weight (in this case base
pairs) on the log scale it is not necessary to take the log of the molecular weight prior to
graphing.
iii. Measure and record the distances migrated by the DNA bands in your PCR reaction. To
determine the size of your DNA bands, first locate the position on the x-axis that indicates the
distance migrated by that band. Then, use a ruler to draw a vertical line from this point to its
intersection with the “standard curve.” Next, extend a horizontal line from this intersection
point to the y-axis. The number on the y-axis is the calculated base-pair size of your PCR
product.
e. It is common to see a second band lower on the gel. This diffuse (fuzzy) band is "primer dimer,"
an artifact of the PCR reaction that results from the primers overlapping one another and
amplifying themselves. Primer dimer is approximately 50 bp, and should be in a position ahead
of the 121 bp marker.
f.
Additional faint bands, at other positions on the gel, occur when the primers bind to chromosomal
loci other than tubulin or 35S and give rise to "nonspecific" amplification products.
2. How would you interpret a lane in which you observe primer dimer, but not the 164 bp or 162 bp
bands?
The presence of primer dimer confirms that the reaction contained all the components necessary for
amplification, but there was insufficient template to amplify target sequences.
Detecting Genetically Modified Food with PCR
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