Download Part III: Laboratory – Electrophoresis

TEXT FOR FRONT PAGE:
In this exercise, the class will map the AGO1 gene of Arabidopsis thaliana through a PCR-based
detection of DNA polymorphisms called CAPS markers (Cleaved Amplified Polymorphic Sequences)
(Konieczy et al, 1993). AGO1 is located somewhere on chromosome 1 and the mutation of both copies
(homozygotes +/+) produces a dwarf plant since the gene expression affects leaf, flower, and auxiliary
meristem development (Bohmert et al, 1998).
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.
 four methods (DNA extraction, Polymerase Chain Reaction (PCR), and gel.
electrophoresis) that are commonly used in biological research.
 about DNA polymorphisms and how they are used in mapping genes.
 about Arabidopsis thaliana, an important research model for molecular genetics.
 about development and cellular differentiation in a multi-cellular organism.
Introduction:
It is often necessary to determine the genetic map position of a gene defined only by a mutation.
Map positions are useful for testing whether a mutation corresponds to a previously identified gene, and
are essential for map-based strategies of gene cloning. Since Alfred Sturtevant’s 1913 mapping
experiments with Drosophila (http://vector.cshl.org/dnaftb/11/concept/index.html), new mutations have
been mapped by linkage analysis. Determining the map position of a gene (as identified by its mutant
phenotype) consists basically of testing the linkage with a number of previously mapped genes or
“markers” that also provide a phenotype. Genetic maps are constructed based on the principle that the
frequency of recombination between genes decreases as the distance between them decreases. The
frequencies of recombination between the gene of interest and the genes previously mapped allow the
gene of interest to be placed on the map.
However, markers for genetic mapping don’t necessarily have to be mutations that cause
phenotypic changes. They can also be variations in DNA sequences that are detectable by molecular
methods. In Arabidopsis thaliana, molecular markers exploit the natural differences between distinct
ecotypes (sub-divisions of species). For instance, it has been estimated that the widely used Landsberg
(Ler) and Columbia (Col) ecotypes differ by approximately 0.5 to 1% at the DNA level. The local
differences or polymorphisms of the DNA sequence are due to point mutations, insertions or deletions that
randomly occurred in one ecotype and not in the other. These DNA polymorphisms can be conveniently
visualized by several methods.
For CAPS mapping, a plant of a certain ecotype (i.e, Ler) that is homozygous for the mutation
ago1 (+/+ for the mutation) is crossed to a wild-type plant (-/-) of a different ecotype (i.e., Col) (see Figure
1). The F1 progeny obtained is heterozygous for the mutation (+/-) and has a chromosome of the Ler
ecotype and a chromosome of the Col ecotype. An F1 plant is allowed to self-fertilize. The resulting F2
progeny is composed of plants that are homozygous wild-type (-/-; about ¼), heterozygous for the
mutation (+/-; about ½), and homozygous for the mutation (+/+; about ¼). Due to crossing-over events
during gamette formation, the chromosomes in the F2 are made of a mixture of the two ecotypes (Ler and
Col).
Figure 1.
Development of the F2 plants needed to test linkage when mapping with CAPS markers.
The star indicates that the gene of interest is mutated at an arbitrary position.
We will take advantage of the mixture of ecotypes in the chromosomes of the F2 progeny to
evaluate the number of crossing-over events between different regions of the chromosome and the gene
AGO1 and thus to locate the gene. The F2 plants that are homozygous for the mutation of interest (+/+),
and thus showing the mutant phenotype, will be used for mapping. Since both of their chromosomes
contain the mutation and the mutation was from a Ler background, the number of crossing-over events is
equivalent to the number of times the Col ecotype is found on the chromosome.
There are many DNA sequence variations among Arabidopsis ecotypes, and since these are also
segregating in the cross, they can be used as genetic markers. Among these variations, CAPS markers
are very useful. They are found in sections of DNA that contain a restriction site present in one ecotype,
but not in another. We will use four CAPS markers located along chromosome 1 (135 cM long) so we can
identify all the sections (see Figure 2):
m235
31.9
cM
2
g4026
84.9 cM
Chr 1
135 cM
UFO
H77224
47.5
113.2 cM
cM
Schematic location of the CAPS markers that will be used on chromosome 1 of
Figure 2.
Arabidopsis thaliana.
Note: The scientific community has generated a large number of CAPS markers. A list is publicly
available through The Arabidopsis Information Resource (TAIR) at the URL:
http://www.arabidopsis.org/aboutcaps.html.
Analysis of CAPS Markers
The CAPS markers are detected using PCR amplification and restriction analysis. The sections
of chromosomes corresponding to the CAPS markers are amplified with specific PCR primers (the
product is the same size for all ecotype DNA). The amplified DNA is then cut by a restriction enzyme. In
the example of Figure 3, the enzyme cuts twice in the Ler ecotype DNA and three times in the Col ecotype
DNA. The results of the restriction are detected by gel electrophoresis. The pattern of the bands will
indicate if the plant is homozygous for the allele from one ecotype (Ler/Ler), heterozygous (Col/Ler), or
homozygous for the allele from the other ecotype (Col/Col), at the position of the CAPS marker.
Figure 3.
Assaying CAPS markers by agarose gel electrophoresis. In this case, the diagnostic
restriction enzyme cleaves the amplified fragment at either two or three sites depending on the ecotype of
Arabidopsis.
Calculating the Recombination Frequency
In this exercise, the recombination frequency (r) between a particular CAPS marker and the gene
of interest is proportional to the number of chromosomes that are Col at the CAPS marker. Its value in %
is obtained by the following formula:
Number of Col/Ler + 2 X Number of Col/Col
r =
X 100
3
2 X Number of plants analyzed
It is necessary to convert the recombination frequency (in %) to a map distance (D, in cM). In
Arabidopsis, a reasonable estimate of map distance is given by the Kosambi function:
D = 25 x ln [ (100 + 2r) / (100 – 2r) ]
REFERENCES
1. Bohmert, K., Camus, I., Bellini, C., Bouchez, D., Caboche, M., and Benning, C. (1998). AGO1 defines a
novel locus of Arabidopsis controlling leaf development. EMBO Journal 17, 170-180.
2. 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.
3. Konieczny A and Ausbel FM (1993) A procedure for mapping Arabidopsis mutations using co-dominant
ecotype-specific PCR-based markers. Plant J. 4: 403-410.
TEXT FOR MATERIALS:
Kits are available through Carolina Biological Supply Company and include the following:
Store in a –20C freezer
1
tube of m235 primer/loading dye mix, 700 L
1
tube of g4026 primer/loading dye mix, 700 L
1
tube of UFO primer/loading dye mix, 700 L
1
tube of H77224 primer/loading dye mix, 700 L
1
tube of HindIII, 25 L
1
tube of RsaI, 25 L
1
tube of TaqI 25 L
1
tube of HindIII resrtriction buffer mix, 240 L
1
tube of RsaI resrtriction buffer mix, 240 L
1
tube of TaqI resrtriction buffer mix, 480 L
1
tube of ready to load marker (pBR322 cut with BstN1) (130 L/9.75 g)
Store at room temperature
15
pellet pestles
100
Ready-To-GoTM PCR beads in either 0.5 mL or 0.2 mL PCR tubes
6
tubes of TE buffer, 1 mL
6
tubes of Edward’s Buffer, 2 mL
6
tubes of isopropanol, 2 mL
2
tubes of arabidopsis seed, 25 seeds
1
seed growing tray
1
dome lid for growing tray
1
planting container with 6 cells
1
bag of soil, 4 cups
1
bottle of mineral oil, 5 mL
1
Teacher’s Guide
6
Students’ Guides
Amplification and Electrophoresis Kits also contain
1
bottle of 20X TBE, 150 mL
8
vials of agarose, 2 g
16
staining trays
4
1
1
1
8
bottle of 1 g/mL ethidium bromide with MSDS sheet, 250 mL (in kits with ethidium
bromide)
bottle of CarolinaBLU final stain, 250 mL (in kits with Carolina BLU )
bottle of CarolinaBLU gel and buffer stain, 7 mL (in kits with Carolina BLU )
latex gloves
Additional equipment needed, but not provided
1.5 mL microcentrifuge tubes (minimum of 36)
microcentrifuge
thermal cycler
water bath (37C)
water bath (65C)
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 3-4 weeks prior to doing the
lab. See instructions for planting and growing seed.
The lab can be broken into three parts:
I.
II.
III.
IV.
V.
Isolating DNA from Arabidopsis tissue using Edward's buffer.
Amplifying the APS markers by PCR.
Analyzing the amplified DNA by agarose gel electrophoresis.
Digestion of CAPS PCR products by restriction enzymes.
Analyzing the restriction digests by agarose 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.
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 Arabidopsis seeds
3-4 weeks
before lab
15-30 min.
Planting Arabidopsis seeds
30 min.
30-60 min.
Pre-lab: Set-up student lab stations
Isolate Arabidopsis DNA
I. DNA Isolation
1
Activity
5
II. PCR Amplification
2
III. Gel Analysis
3
4
IV. Restriction Digest
5
V. Gel Analysis
6
7
30-60 min.
15-30 min.
70+ min.
30 min.
30 min
30+ min.
20+ min.
20 min. to O/N
20 min.
30-60 min.
15-30 min.
60+ min.
30 min.
30 min
30+ min.
20+ min.
20 min. to O/N
20 min.
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
Pre-lab: Set-up student lab stations. Aliquot reagents
Set-up restriction digest reactions
Post-lab: Incubate in water bath
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
Planting and Growing Seed
Plant Arabidopsis seeds as described below and allow for a 3-4 week growth period. Depending
upon growing conditions, you may see the difference in phenotype between the different plants as early as
2 weeks. For futher information concerning growing Arabidopsis, refer to The Arabidopsis Information
Resource (TAIR) at www.arabidopsis.org.
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
Arabidopsis seed.
2.
Plant the seeds by scattering them evenly on top of the soil. This is not as simple as it appears and
should be done carefully. The seeds are very tiny and difficult to handle. Scatter the seeds using an
approximately 4 inch by 4 inch sheet of paper folded in half. Place the seeds into the fold of the paper
and gently tap them onto the soil. Plant each of the two groups of seeds included with the kit
separately. Make sure that the seeds are scattered 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 and cover with the plastic
dome lid. Water the plants by adding one-quarter inch of water to the growing tray. You may wish to
keep a small amount of water in the growing tray at all times 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 under 24 hours of cool, white fluorescent light at 20-22C (room temperature) to be
able to discern the difference in phenotype as early as two to three weeks. If the plants are grown
under 16 hours of light and 8 hours of dark (the more traditional growing conditions), it may take
longer to discern the different phenotypes. You may need to place the plants directly underapproximately one foot-the lights to achieve optimum growth. The clear plastic dome may be left over
the plants to retain moisture.
5.
Harvest plant tissue for PCR as soon as the difference in phenotype between the plants becomes
obvious. This should be about 2-4 weeks after planting (before they flower), and will depend upon the
light and temperature conditions. As described in the introduction, the plants that are homozygous for
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the ago-1 mutation have a dwarf phenotype with serrated leaves. The most obvious characteristic of
these plants is the decrease in size in comparison to the wild type and heterozygous plants. The
mutant plants are very small. 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.
TEXT FOR METHODS
PART I: ISOLATING DNA FROM ARABIDOPSIS THALIANA
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
1. Obtain an Arabidopsis plant and observe and record its phenotype. Take a piece of leaf tissue that is
approximately an eighth of an inch in diameter. If the leaves are too small, take tissue from multiple
leaves (from the same plant) until you have the equivalent amount of leaf tissue. Note: Plants with the
ago-1 phenotype are so small that you may have to use the entire plant. If you use the entire plant,
make sure that no soil remains clinging to the roots. Place the leaf tissue in one of the microcentrifuge
tubes on your bench.
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.
7
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.
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. DNA can be used immediately 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 four PCR reactions to analyze each plant. Each PCR reaction amplifies a
specific CAPS marker on chromosome 1 of Arabidopsis. Therefore you will use four different sets of
primers: m235, g4026, UFO, and H77224. Each CAPS marker primer set amplifies a PCR product with a
specific length in base pairs. The table below displays the expected PCR product sizes for each primer
set.
CAPS Marker Primer Set
m235
g4026
UFO
H77224
PCR product size (bps)
534
800
1299
220
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.
8
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.
Thermal Cycling
PCR amplification from crude cell extracts is biochemically demanding, and requires the precision of
automated thermal cycling. However, amplification of the CLF 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, 65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
Primer/loading dye mix for each
CAPS marker (4), 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
1.
For each CAPS marker, use a micropipet with a fresh tip to add 22.5 l of CAPS marker primer
mix to a PCR tube containing a Ready-To-Go PCR Bead. Tap tube with finger to dissolve bead.
Make sure to label the four tubes to know which CAPS marker will be amplified in each of the
tubes.
2.
Use a fresh tip to add 2.5 µL of Arabidopsis 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.
3.
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.
4.
Store all samples on ice 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.
9
Denaturation step: Time Temp
Annealing step: Time – Temp
Extension step: Time – Temp
5.
30 sec - 94C
30 sec - 65C
30 sec - 72C
Store the DNA amplified through PCR at -20°C until you are ready for the gel analysis and the
enzymatic restriction.
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
Needed, not Supplied
10
Shared Items
pBR322/BstNI markers
Staining tray
2.0 - 20 L micropipet and
tips
20 – 200 L micropipet and
tips
2% agarose in 1X TBE*
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. Use a micropipet with a fresh tip to transfer the 5 l of each of the four sample/loading dye
mixtures into your assigned wells 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. Visualize the results. You are expecting the following bands: for m235 a band at 534 bp, for UFO
a band at 1300 bp, for g4026 a band at 900 bp and for H77224 a band at 220 bp.
11
Staining with CarolinaBLU
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
12
Procedure IV. Cutting the DNA amplified by PCR with restriction enzymes.
PART IV: Pre-lab Notes
Important Note
This procedure uses restriction enzymes to determine the allelic ecotype for each of the each
amplified CAPS markers. To score the alleles, you will need to set up four different restriction digests of
the PCR products of each of the CAPS markers- m235, g4026, UFO, and H77224. The table below
displays the restriction enzyme specific for each CAPS marker PCR product.
CAPS Marker Primer Set
m235
g4026
UFO
H77224
Restriction Enzyme
HindIII
RsaI
TaqI
TaqI
The expected CAPS restriction fragment lengths, for each ecotype, are listed in the table below.
CAPS Marker
m235a
UFOa
g4026a
H77224a
Col
309 + 225 bp
983 + 316 bp
650 bp
130 + 90 bp
Ler
534 bp
600 + 383 + 316 bp
800 bp
130 + 70 + 20 bp
Restriction enzyme/Buffer mix
For each CAPS marker a restriction enzyme /buffer mix can be made ahead of time. This mixture
contains 1 L of 10X BSA, 1L of appropriate 10X restriction Buffer, 7 L of distilled water and 1L of the
appropriate enzyme. This can be scaled up to prepare for 26 reactions and aliquoted ahead of time. The
restriction enzyme/buffer mix can be stored on ice for several hours before use. Do not freeze, as this
destroys the enzyme activity.
Setting Up Restriction Digests
Students should obtain four new i.5 ml microfuge tubes and label them m235, g4026, UFO, and
H77224. Students add the appropriate CAPS PCR product to the appropriately labeled reaction tubes.
The students then add 10µL of the appropriate restriction enzyme/buffer to the CAPS PCR products.
Make sure the student’s digests are incubated in the proper temperature for the restriction enzyme used.
Set-up Each Student Lab Station With:
From Kit or Part I
PCR product of each CAPS marker
(4), 12 L (on ice)
Restriction enzyme/Buffer mix for
each CAPS marker (4) 12 L (on
ice)
Shared Items
Needed, Not Supplied
1-20 µL micropipet and tips
Water bath (37C)
20-200 L micropipet and tips
Water bath (65C)
Ice bucket
PROCEDURE
1.
Transfer 10 l of amplified DNA from each CAPS marker to a fresh tube. You should label the
tubes: m235, UFO, g4026, H77224.
2.
Add 10 l of the appropriate restriction enzyme/ buffer mix to the 10 l of the PCR product. See
the table below for restriction enzymes and conditions.
m235
HindIII/buffermix
cuts at 37C
13
UFO
TaqI/buffer mix
g4026 RsaI/buffer mix
H77224 TaqI/buffer mix
cuts at 65C
cuts at 37C
cuts at 65C
3.
Vortex the samples briefly to mix.
4.
Place your tubes in the appropriate water bath (37C or at 65C) and incubate for at least 1 hour.
5.
The digestions can be stored at -20C until you are ready to do the gel analysis.
PART V: ANALYZING THE RESTRICTION DIGESTS BY GEL ELECTROPHORESIS
Part V: 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 V: 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:
14
Shared Items
From Kit
pBR322/BstNI markers
Staining tray
Needed, not Supplied
2.0 - 20 L micropipet and
tips
20 – 200 L micropipet and
tips
2% agarose in 1X TBE*
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 V: 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. Use a micropipet with a fresh tip to transfer the 5 l of each of the four sample/loading dye
mixtures into your assigned wells 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. Visualize the results. You are expecting the following bands: for m235 a band at 534 bp, for UFO
a band at 1300 bp, for g4026 a band at 900 bp and for H77224 a band at 220 bp.
15
Staining with CarolinaBLU
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
16
PROCEDURE
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. Add and mix 4 l of cresol red loading dye to each restriction digest. Make sure to change tips
each times.
5. Use a micropipet with a fresh tip to transfer the 20 l of sample/loading dye mixture 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).
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 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.
9. Photograph gels.
17
RESULTS AND DISCUSSION
1. Visualize the results. You are expecting the following bands for each CAPS marker and each
ecotype of Arabidopsis:
CAPS Marker
m235a
UFOa
g4026a
H77224a
Col
309 + 225 bp
983 + 316 bp
650 bp
130 + 90 bp
Ler
534 bp
600 + 383 + 316 bp
800 bp
130 + 70 + 20 bp
2. 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. Use the sample gels pictured below to help you.
UFOa
m235a
g4026a
H77224a
3. Compile the results of the four CAPS markers to locate the AGO1 gene on a map of chromosome
4. The recombination frequency (r) between a particular CAPS marker and the gene of interest is
proportional to the number of chromosomes that are Col at the CAPS marker. Its value in % is
obtained by the following formula:
Number of Col/Ler + 2 X Number of Col/Col
18
r =
X 100
2 X Numner of plants analyzed
5. Look at all the results the class obtained and evaluated the percentage of recombination of each
CAPS marker with the AGO1 gene.
6. It is necessary to convert the recombination frequency (in %) to a map distance (D, in cM). In
Arabidopsis, a reasonable estimate of map distance is given by the Kosambi function:
1. D = 25 x ln [ (100 + 2r) / (100 – 2r) ]
7. Convert the percent of recombination obtained between each CAPS marker and the AGO1 gene
into map distance. Use the following map to locate the position of the AGO1 gene on
chromosome 1.
m235a
31.9
cM
Chr 1
135 cM
g4026a
84.9 cM
UFOa
47.5
cM
H77224a
113.2 cM
19