Download Part III: Laboratory – Electrophoresis

TEXT FOR FRONT PAGE:
Analysis of Genotype/Phenotype using the bronze-2 mutation in maize
This laboratory uses a rapid method to isolate DNA from plant tissue for genotyping maize by
using the Polymerase Chain Reaction (PCR) method (Edwards, et al., 1991). You will amplify a small
region of the BRONZE (BZ) gene to identify the genotype of the plant with respect to the gene. Maize is
diploid, so a single plant contains two copies of the BZ gene. Thus, there are three possible genotypes
with respect to the CLF gene for any given plant: homozygous-mutated/mutated, heterozygousmutated/wild type, and homozygous-wild type/wild type. If the gene is mutated, amplification of the locus
will produce a 750 bp product. If the gene is in the wild type state, amplification of the locus will produce a
smaller 250 base pair product. Once PCR has been performed, the genotype of a given plant is
determined by using agarose gel electrophoresis to analyze the base pair lengths of the PCR products
amplified from the BZ locus. The genotype can then be compared to the phenotype of the plant from
which the DNA was isolated. The BZ gene encodes a glutathione S-transferase that performs the last
genetically defined step in the maize anthocyanin biosynthesis pathway. Mutations to this gene lead to the
accumulation of an anthocyanin precursor that confers a bronze pigmentation onto the ears and stalk of
corn.
TEXT FOR PRELAB NOTES:
Objectives/Goals:
This laboratory can be used to teach students:
 the molecular basis of heredity.
 the relationship between genes, proteins, and traits.
 basic Mendelian Genetics
 three methods (DNA extraction, Polymerase Chain Reaction (PCR), and gel
electrophoresis) that are commonly used in biological research.
 about transposable elements, a powerful tool for basic research.
 about Zea mays (maize), an important research model for molecular genetics
 about development and biochemical pathways in a multi-cellular organism
Introduction:
The Discovery of “Jumping Genes”
In the mid 1940's most geneticists assumed the genome was a static entity with stable genes,
replicating faithfully as cells divided and organisms developed. Observations made by Barbara McClintock
at Cold Spring Harbor Laboratory told a radically different story. McClintock observed that regions of DNA
could jump, or "transpose". This observation challenged the simplistic view of how a genome was
supposed to work. McClintock's transposable DNA elements, popularly known as "jumping genes", offered
an explanation to gene expression patterns in plants that Mendel's Laws failed to provide. However, her
work was not immediately accepted by many of her fellow researchers. It took the development of
recombinant DNA methods and the discovery of transposable elements in everything from bacteria to
humans, to give her ideas wide acceptance. In 1983, three decades after her work was first published,
she was awarded the Nobel Prize for her work. Currently, transposable elements are one of the most
powerful tools in the study of plant growth and development.
McClintock's first clue to the existence of mobile DNA elements came from studies of a maize
locus where chromosomal breakage occurred (McClintock, 1951). She called this region of chromosomal
breakage "Dissociator” (Ds). For the Ds element to break from the chromosome, it required the presence
of a second element that she called "Activator" (Ac). Using a classical genetics approach, McClintock was
able to demonstrate how Ac and Ds interacted to affect gene expression. In maize, the wild type corn
kernel is purple. McClintock observed that Ds was not at its normal chromosome location in a strain of
maize that possessed a kernel pigment mutation. Unable to produce the pigment anthocyanin, this mutant
strain produced ears of corn with white kernels instead of purple kernels. When the mutant strain was
crossed to strains containing Ac, Ds transposition caused the wild type kernel color to be restored,
producing purple speckles on a white kernel background. To McClintock, this suggested Ds transposition
could inactivate gene expression by jumping into genes or reactivate gene expression by jumping out of
genes. Furthermore, McClintock observed that Ac has the ability to transpose and cause insertion
mutations. Unlike Ds though, Ac has the ability to jump independently. She hypothesized that independent
mobility of Ac would explain the instability associated with Ac mutations. Due to their ability to control gene
expression, she referred to Ac and Ds as “Controlling Elements”. Years later, her description of the Ac/Ds
system would serve as a model to explain the speckled and variegated gene expression patterns so
common to plants.
The BRONZE (BZ) Gene
This lab will analyze the BRONZE (BZ) gene of maize to demonstrate the molecular relationship between
genotype and phenotype. The Bronze-2 gene encodes a glutathione S-transferase that performs the last
genetically defined step in the maize anthocyanin biosynthesis pathway. Mutations to this gene lead to the
accumulation of an anthocyanin precursor that confers a bronze pigmentation onto the ears and stalk of
corn. Wildtype plants have red/purple anthocyanins located in the cell vacuole, whereas bz-2 mutants
have a tan/bronze color dispersed throughout the cell cytosol (Dooner, et al., 1985).
Place pictures of the bronze and WT maize corn here.
REFERENCES
1. 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.
2. McClintock, B. (1951). Chromosome Organization and Genic Expression. Cold Spring Harbor Symp.
Quant. Biol. 16:13-47.
3. Dooner, H. K., E. Weck, S. Adams, E. Ralston, M. Favreau et al., (1985) A molecular genetic analysis
of insertion mutations in the bronze locus in maize. Mol. Gen. Genet. 200:240-246
TEXT FOR MATERIALS:
Kits are available through Carolina Biological Supply Company and include the following:
Store in a –20C freezer
1
tube of bz1/bz2 primer/loading dye mix, 700 L
1
tube of bz1/Ds primer/loading dye mix, 700 L
1
tube of ready to load marker (pBR322 cut with BstN1) (130 L/9.75 g)
Store at room temperature
15
pellet pestles
25
Ready-To-GoTM PCR beads in either 0.5 mL or 0.2 mL PCR tubes
2
6
6
6
2
1
1
1
1
1
6
tubes of TE buffer, 1 mL
tubes of Edward’s Buffer, 2 mL
tubes of isopropanol, 2 mL
tubes of maize seed, 25 seeds
seed growing tray
planting container with 6 cells
bag of soil, 4 cups
bottle of mineral oil, 5 mL
Teacher’s Guide
Students’ Guides
Amplification and Electrophoresis Kits also contain
1
bottle of 20X TBE, 150 mL
4
vials of agarose, 2 g
6
staining trays
1
bottle of 1 g/mL ethidium bromide with MSDS sheet, 250 mL (in kits with ethidium
bromide)
1
bottle of CarolinaBLU final stain, 250 mL (in kits with Carolina BLU )
1
bottle of CarolinaBLU gel and buffer stain, 7 mL (in kits with Carolina BLU )
4
latex gloves
Additional equipment needed, but not provided
1.5 mL microcentrifuge tubes (minimum of 36)
microcentrifuge
thermal cycler
equipment for gel electrophoresis
pipets and sterile tips (for measuring volumes from 2.5 L to 400 L)
ice buckets with crushed ice
vortex (optional)
white light box (to visualize DNA with CarolinaBLU) (optional)
UV transilluminator (to visualize DNA with ethidium bromide)
microcentrifuge tube racks (can use empty pipet tip boxes)
permanent markers
TEXT FOR PRELAB PREP
Lab Flow and Summary
Note: You must plant the arabidopsis seed that comes with the kit 1-3 weeks prior to doing the
lab. See instructions for planting and growing seed.
The lab can be broken into three parts:
I.
II.
III.
Isolating DNA from maize tissue using Edward's buffer.
Amplifying the CLF locus by PCR. During this step, specific primers are used to analyze both
possible alleles-the wild type and the Ds created mutant.
Analyzing the amplified DNA by gel electrophoresis.
The laboratory is organized by part. Each part has notes for the instructor (when necessary),
preparation instructions, and the experimental protocol for the lab. A separate Results and Discussion is
also provided with some additional suggested analysis.
The lab is set-up to have students work in pairs and for each lab station to have two pairs of
students working there and sharing one gel. Each pair of students will work together to extract the
DNA from which two separate PCR reactions will be set-up. One student in the pair will set-up one PCR
reaction and the other will set up the other PCR reaction.
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The following table will help you plan and integrate the three parts of the experiment. Remember,
you will need to plant the Arabidopsis seed that comes with the kit 3-4 weeks ahead of the actual
lab. Specific instructions for planting and growing the seed are included.
Part
Day
Time
Plant maize seeds
1-3 weeks
before lab
15-30 min.
Planting maize seeds
30 min.
30-60 min.
30-60 min.
15-30 min.
70+ min.
30 min.
30 min
30+ min.
20+ min.
20 min. to O/N
20 min.
Pre-lab: Set-up student lab stations
Isolate maize DNA
Pre-lab: Set-up student lab stations. Aliquot reagents
Set-up PCR reactions
Post-lab: Amplify DNA in thermal cycler
Prepare agarose gel solution and cast gels
Load DNA samples into gels
Electrophoresis
Post-lab: Stain gels
Post-lab: De-stain gels
Post-lab: Photograph gels
I. DNA Isolation
1
II. PCR Amplification
2
III. Gel Analysis
3
4
Activity
Planting and Growing Seed
Plant maize seeds as described below and allow for a 1-3 week growth period. Depending upon
growing conditions, you may see the difference in phenotype between the different plants as early as 1
week.
1.
Pre-wet the soil that comes with the kit and fill the 6 small cells that make up the planting container.
Note: You must use the soil that comes with the kit. It has been found to work well for growing maize
seed.
2.
Plant the seeds 0.5 inches below the soil using your finger. Plant each of the two groups of seeds
included with the kit separately. Make sure that the seeds are planted with space in between them,
so they will grow better, and their phenotype will be more readily observable.
3.
Place the planting container into the growing tray included with the kit. Water the plants from above
to prevent the soil from drying out. However, do not allow the soil to remain soggy, or your seeds and
plants will rot.
4.
Grow the plants on close to a sunny window at room temperature or slightly warmer. If the plants are
grown under cold conditions, it may take longer to discern the different phenotypes.
5.
Harvest plant tissue for PCR as soon as the difference in phenotype between the plants becomes
obvious. This should be about 1-3 weeks after planting and will depend upon the light and
temperature conditions. As described in the introduction, the plants that are homozygous for the bz-2
mutation have bronze pigmented stalks and seeds. The most obvious characteristic of these 1-3
week old plants is the bronze stalk when compared to the wild type and heterozygous plants. You
may wish to allow the plants to continue to grow after you have harvested the plant tissue as the
phenotypic difference between the homozygous mutant plants and the wild type and heterozygous
plants becomes more obvious with time.
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TEXT FOR METHODS
PART I: ISOLATING DNA FROM MAIZE
PART I: PRE-LAB SET-UP
Two student pairs wil work at each lab station
Set-up Each Student Lab Station With
From Kit
Edward's Extraction Buffer,
2 mL tube
Needed, Not Included with Kit
4-1.5 mL microcentrifuge tubes,
polypropylene
Isopropanol, 2 mL tube
100-1000 µL micropipet and tips
Shared Items
Microcentrifuge
Arabidopsis plants
Tris/EDTA (TE) Buffer, 1 mL tube
2 Disposable pellet pestles
PART I: LABORATORY-ISOLATING DNA
PROCEDURE
1. Grind tissue in a microfuge with plastic pestle for 1 minute. Note: It is important to use tissue from
young plants. The quantity is also important. Best results come from using the opposite end of a
1000 µl micropipette to “punch” a whole in the leaf. The resulting leaf disk is the ideal size.
2. Grind the plant tissue forcefully in the microcentrifuge tube using the plastic pellet pestle. Grind for
approximately 1 minute. The sample should look like green liquid when it is fully ground.
3. Add 400 L of Edward's Extraction Buffer to the ground plant tissue.
4. Grind briefly (to remove tissue from the pellet pestle and to liquify any remaining pieces of tissue).
5. Vortex the tube for 5 seconds; leave at room temperature for 5 minutes.
6. Microcentrifuge the tube containing the ground plant tissue and Edward’s buffer for 2 minutes. After 2
minutes any insoluble plant tissue should form a tight pellet at the bottom of the tube.
7. Transfer 350 L of the supernatant to a fresh tube. This supernatant contains the desired DNA. Make
sure not to disturb the pelleted plant tissue when transfering the supernatant.
8. Add 400 L of isopropanol to the DNA containing supernatant, mix, and leave at room temperature for
3 minutes. This step is to precipitate the DNA.
9. Microcentrifuge the tube with the isopropanol and supernatant for 5 minutes with the hinge of the tube
against the back wall of the rotor. Carefully remove the supernatant completely. The pellet should be
located on the side that was against the back wall of the centrifuge (the side with the hinge) during the
spin. It will be small and may be very difficult to see. Air dry the pellet for 10 minutes to remove any
remaining isopropanol.
5
10. After drying, resuspend the DNA pellet in 100 l of TE Buffer.
11. Centrifuge the DNA in TE for 1 minute to pellet any plant material that did not go into solution. You will
use 2.5 L of this supernatant as the template DNA for the PCR reactions.
12. The DNA can be used immediatly or stored at -20C. During use keep the DNA on ice.
PART II: AMPLIFYING DNA BY PCR
PART II: Pre-lab Notes
Important Note
This procedure uses two PCR reactions to analyze each plant. Since the Ds element insertion
creating the bz-2 mutation is so large, detecting the mutation by amplifying across the Ds element is
impractical. This lab uses an alternative approach involving 2 distinct primer sets. One primer set (bz1/Ds)
contains a primer that anneals to the BZ gene and a primer that anneals to the Ds element at the mutated
bz-2 locus. This primer set amplifies only the mutated allele. The second primer set (bz1/bz2) amplifies
the wild type BZ allele. If the plant demonstrates a normal phenotype, it should have a least one copy of
the wild type allele. Therefore, the primer set amplifying the wild type allele 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 Ds element and the accompanying mutation and not of a problem with template
quantity or quality. The PCR products from the two primer sets are different in size- 750 bp for the bz1/Ds
primer set and 250 bp for the bz1/bz2 primer set. This allows for easy resolution of the two alleles by 2%
agarose gel electrophoresis.
Include the figure showing the primer location here.
Ready-To-Go PCR BeadsTM
Each PCR bead contains reagents so that when brought to a final volume of 25 L the reaction
contains 1.5 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 in Tris-low EDTA (TLE) buffer (10mM Tris-HCl, pH 8.0; 0.1 mM EDTA).
Setting Up PCR Reactions
The lyophilized Taq polymerase in the Ready-To-Go PCR Bead becomes active immediately upon
addition of the primer/loading 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, and initiate thermal cycling as soon as possible after mixing the PCR
reagents. Be sure the thermal cycler is set and have all experimenters set up their PCR reactions
coordinately. Add primer/loading dye mix to all reaction tubes, then add each student template,
and begin thermal cycling immediately.
To insure maximum specificity, some experimenters employ a "hot start" technique where one reagent
is withheld from the reactions until the samples are cycled to the initial denaturing temperature. You can
perform a hot start by adding the DNA template during the first denaturation step. Either program an
extended first denaturation of 10 minutes, or stop cycling and restart after adding template. A simpler
alternative is to set up the reactions on ice, start the thermal cycler, and then place the tubes in the
machine as the temperature approaches the denaturing set point.
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Thermal Cycling
PCR amplification from crude cell extracts is biochemically demanding, and requires the precision of
automated thermal cycling. However, amplification of the BRONZE locus 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, 58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 30 cycles.
PART II: PRE-LAB SET-UP
Two student pairs will work at each lab station
Note: You may wish to aliquot the primers into 24 L aliquots so that each student pair has their own
supply of primers.
Set-up Each Student Lab Station With:
From Kit or Part I
2 aliquots primer/loading dye mix
bz1/bz2, 24 L (on ice)
2 aliquots primer/loading dye mix
bz1/Ds, 24 L (on ice)
Student isolated Arabidopsis DNA
(on ice)
4 Ready to go PCR Beads (in
reaction tubes)
Shared Items
Needed, Not Supplied
1-20 µL micropipet and tips
Thermal cycler
20-200 L micropipet and tips
Mineral oil (depending on thermal
cycler used)
Ice bucket
PART II: LABORATORY - AMPLIFYING ARABIDOPSIS DNA BY PCR
PROCEDURE
1.
Use a micropipet with a fresh tip to add 22.5 µL of the bz1/bz2 primer/loading buffer mix to a PCR
tube containing a Ready-To-Go PCR Bead. Tap the tube very gently with your finger to dissolve
the bead. Label the tube appropriately.
2.
Use a micropipet with a fresh tip to add 22.5 µL of the bz1/Ds primer/loading buffer mix to a
second PCR tube containing a Ready-To-Go PCR Bead. Tap the tube very gently with your finger
to dissolve the bead. Label the tube appropriately.
3.
Use a fresh tip to add 2.5 µL of Maize DNA (from Part I) to each reaction tube. Mix by gently
pipetting 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.
Add one drop of mineral oil to the top of the reactants in the PCR tube. 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.
5.
Store all samples on ice or in the freezer until you are ready to amplify according to the following
program. Program the thermal cycler for 30 cycles according to the following cycle profile. The
program may be linked to a 4°C hold program to hold the samples at 4°C after the 30 cycles are
completed.
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Denaturation step: Time Temp
Annealing step: Time – Temp
Extension step: Time – Temp
30 sec - 94C
30 sec - 58C
30 sec - 72C
PART III: ANALYZING AMPLIFIED DNA BY GEL ELECTROPHORESIS
Part III: Pre-Lab Notes:
Loading and Electrophoresing Samples
The object in these experiments 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 functions as loading dye, so that amplified samples
can be loaded directly into gels. 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 produces fragments that are
useful as size markers in this experiment and has been included with the kit. 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 µL of the DNA
ladder per gel.
Viewing and Photographing Gels
View and photograph gels as soon as possible after the appropriate destaining. Over time,
especially if you have stained with ethidium bromide, PCR products no longer appear as stained bands as
they slowly diffuse through the gel. If kept refrigerated and in a very small amount of distilled or deionized
water, CarolinaBLU stained gels will retain their integrity for months.
Part III: 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.
Note: You may wish to aliquot the marker so that each student station has its own tube.
Set-up Each Student Lab Station With:
From Kit
pBR322/BstNI markers
Shared Items
Needed, not Supplied
2.0 - 20 L micropipet and
tips
8
2% agarose in 1X TBE*
Staining tray
20 – 200 L micropipet and
tips
1X TBE buffer*
Electrophoresis chamber
Mineral oil (in kit)
Electrophoresis power
supply
1 mg/mL ethidium bromide
or CarolinaBLU staining
solutions*
2-1.5 mL microcentrifuge
tubes
Transilluminator w/camera
* Included with, or materials for, included with some kits.
Part III: Laboratory – Electrophoresis:
1. Prepare a 1X concentration of TBE by adding the contents of the bottle of 20X concentrated stock (150
mLs) to 2850 mL of deionized or distilled water. Mix thoroughly.
2. Seal the ends of the gel tray with masking tape and insert the comb. Prepare a 2% agarose gel in 1X
TBE as follows. Add 8 g of agarose to 400 mL of 1X TBE and heat in a boiling water bath
(approximately 15 minutes), on a hotplate, or in a microwave (approximately 5-10 minutes) until the
agarose is completely dissolved. You should no longer see agarose particles floating in solution. Allow
the agarose to cool so that you can touch the container without burning yourself before pouring it into
the gel tray (55-65C). (If boiling hot agarose is poured into the gel trays without cooling to the touch it
shortens the lifetime of the gel trays.) When the agarose has cooled, pour it into the tray to form a gel
approximately one quarter inch thick. Allow the gel to solidify completely. The gel should be cloudy
when it is completely solidified. This takes at least 20 minutes.
3. Place the gel into the gel rig and cover it with 1X TBE buffer.
4. Combine both PCR products (bz1/bz2 and bz1/Ds) into a fresh 1.5 mL test tube. If you used mineral
oil, either remove the mineral oil prior to doing this, or place your pipet tip below the mineral oil to pipet
up the PCR products and leave the mineral oil behind in the original tube. Pipet up and down to mix.
Use a micropipet with a fresh tip to add 30 µL of the PCR sample/loading dye mixture of the two
reactions into your assigned well of a 2% agarose gel. (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).
5. Load 20L of the molecular weight marker (pBR322/BstN1) into one well.
6. 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.
7. Once the loading dye has run the appropriate distance into the gel, stain the gels by soaking them in
stain. If you are using ethidium bromide, stain for 15 minutes. If you are using CarolinaBlu, see the
instructor for instructions. 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.
8. Photograph gels
Staining with CarolinaBLU
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To stain gels following electrophoresis cover the gel with the Final CarolinaBLU stain and let sit
for 20-30 minutes. Agitate gently, if possible (optional). Pour the stain back into the bottle to be used
another time. (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 a small
volume of water-just enough 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 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 Carolina 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 LEADS TO PRECIPITATION OF THE DNA IN
THE WELLS AND CAN CREATE ARTIFACTIAL AGGREGATED DNA BANDS IN THE AGAROSE GEL.
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 gel and buffer stain to the 1X TBE buffer.
Voltage
Buffer Volume
Stain volume
500 mL
480 L (12 drops)
3 liters
2.88 mL (72
drops)
500 mL
960 L (24 drops)
2.6 liters
5 mL (125 drops)
<50 Volts
>50 Volts
TEXT FOR RESULTS AND DISCUSSION
1.
Observe the photograph of the stained gel containing your sample and those from other students.
Orient the photograph with the sample wells at the top. Interpret the band(s) in each lane of the gel.
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Use the sample gel pictured below to help you.
750 bp, bz1/Ds
250 bp, bz1/bz2
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. 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).
c.
The amplification product of the mutant bz-2 allele (750 bp), which has been amplified using the
bz1/Ds primers, should align between the 929 bp marker and the 383 bp marker. The
amplification of the wild-type allele (250 bp), which has been amplified using the bz1/bz2 primers,
should align between the 383 bp marker and the 121 bp marker.
d. Establish the exact size of your PCR products as follows.
- 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 size of the DNA
fragments in your marker are as follows:1857 bp, 1058 bp, 929 bp, 383 bp, and 121 bp.
- 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.
- 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
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intersection point to the y axis. The number on the y-axis is the calculated base-pair size
of your PCR product.
d. 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 the bz-2 allele and give rise to "nonspecific" amplification products.
.
2. How would you interpret a lane in which you observe primer dimer, but not the 750 bp or 250 bp
bands?
The presence of primer dimer confirms that the reaction contained all the components necessary for
amplification, but that there was insufficient template to amplify the 750-bp and/or 250-bp target
sequence.
3. Based on analysis of the agarose gel, what is the genotype of your plant?
If you observe only the 250 bp band, the plant is homozygous for the wild-type allele. If you observe
only the 750 bp band the plant is homozygous for the mutant bz-2 allele. If you observe both bands,
the plant is heterozygous for the bz-2 allele.
4. Would you classify the bz-2 mutation as recessive or dominant? Explain your reasoning.
The bz-2 mutation is recessive. Plants that are heterozygous do not demonstrate the phenotype. Only
plants that are homozygous for the bz-2 mutation demonstrate the curly leaf phenotype.
5.
Based on the genotypic distribution of the plants analyzed by the class, what genotype was the
parental plant? Why may the genotype distribution observed by your class deviate from what is
expected under Mendel’s Laws of inheritance?
You may or may not wish to make your students aware of the following facts, depending upon
how you want to have them approach answering the question. The seeds for this experiment came
from a parental plant that was heterozygous.
To answer the question have your students count the number of plants homozygous for the wild
type allele, the number of plants that are heterozygous, and the number of plants homozygous for the
mutated clf-2 allele. They should exist in a ratio of approximately 1:2:1. Therefore, the parental plant
was heterozygous.
To increase your students understanding of why this ratio allows them to determine that the
parent is a heterozygote, have them draw Punnett squares showing the three possible self pollination
events. Additionally, you could have them draw Punnett squares for the 6 possible crosses that could
result from outcrossing.
If the distribution deviates significantly, small sample size is the most probable cause. Even if the
ratio doesn’t indicate that the parent plant is heterozygous, if you have any plants that are
heterozygous, the parental plant could only be heterozygous.
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