southern hybrid experiment teacher
TRANSCRIPT
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21-1215
Southern HybridizationExperiment Kit
Teachers Manual
400 ml
Paper towels
Nitrocellulose filteror nylon membrane
Whatman 3MMpaper wicks
Transfer buffer
Weight (500 g)
Glass plate
Whatman 3MM paper
Gel
Support(inverted casting tray)
Plastic wrap
World-Class Support for Science & Math
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This kit contains materials for six groups to perform Southern transfer and
hybridization analysis using the included lambda DNA samples and
biotinylated probe. The intellectual objective of the experiment is to
determine the region of the genome from which the probe sequence is
taken. Southern hybridization analysis requires several steps. Consult thismanual to plan your laboratory sessions. Some of the introduction to this
kit was excerpted with permission from Recombinant DNA and
Biotechnology: A Guide for Teachersby Kreuzer and Massey (2001; ASM
Press, Washington, DC; Carolina Biological Supply catalog #RN-21-2218).
2004 Carolina Biological Supply Company Printed in USA
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Introduction
During many procedures, including DNA library screening, DNA-based
disease diagnosis, and DNA fingerprinting, it is important to know whether
a specific DNA sequence is present in a DNA sample and where it is located
with respect to restriction enzyme sites. Restriction enzyme digestion,
electrophoresis, and staining allow us to cut DNA molecules into
reproducible pieces and to determine the size of these pieces. However,
restriction enzyme analysis alone does not provide information about DNA
sequences present within the fragments. Southern hybridization analysis
combines restriction enzyme analysis and hybridization analysis to provide
this kind of information. Combining these techniques reveals which
fragments from a restriction digest (if any) contain a specific DNA sequence.
In brief, hybridization analysis involves separating (denaturing) the strands
of the DNA molecules to be analyzed and then mixing those separated
strands with many copies of a single-stranded DNA or RNA molecule,
called a probe. The probe contains a sequence complementary to the
nucleotide sequence of interest. When a probe is mixed with single-
stranded (denatured) DNA under the right conditions, hydrogen bonds
form between the probe and its complementary sequence in the DNA
sample being analyzed. The formation of hydrogen bonds between two
complementary strands to create a double-stranded complex is called
hybridization, or annealing. When a DNA or RNA probe bonds to its
complementary sequence in the DNA being analyzed, the probe is said to
be annealing (or hybridizing) to the sample DNA. Hybridization analysis
can be performed either in solution or with one component attached to
some kind of solid support. For Southern hybridization analysis, the
sample DNA is attached to a solid support, such as a nitrocellulose or
nylon membrane.
Hybridization involves several steps. First, the probe and sample DNA are
allowed to hybridize under the appropriate conditions. The correct
temperature, incubation times, and buffer conditions must be used. Next,
the sample DNA is washed using conditions that will remove unhybridized
probe but not the hybridized probe. Finally, the sample DNA is tested for
the presence of the hybridized probe. The probe is labeled with a
radioactive molecular tag (or some other tag) that allows it to be detected
following hybridization.
To begin Southern hybridization analysis, the sample DNA is digested with
restriction enzymes and the resulting fragments are separated by agarose
gel electrophoresis. The DNA must then be transferred from the agarose
gel to a solid support prior to hybridization. In 1975, a scientist named
Southern published a method for transferring DNA fragments from an
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agarose gel to a membrane in a manner that preserved the arrangement of
the fragments as they existed in the gel. Because of the scientists name,
this transfer method is known as Southern transfer or Southern blotting.
To perform Southern transfer, the agarose gel is first soaked in a basicsolution to denature the DNA fragments. After an additional soaking step
to neutralize the base, the gel is placed on a long piece of blotting paper
with the ends of the paper suspended in a reservoir of salt solution. A
nitrocellulose or nylon membrane is then laid directly on top of the gel.
Blotting paper and a stack of dry absorbent paper (such as paper towels)
are then placed on the membrane (see Fig. 2, page 14). The blotting paper
acts like a wick. Driven by capillary action, fluid is drawn from the reservoir
up though the gel and into the stack of dry paper.
As the fluid migrates up through the gel, it carries the denatured DNA
fragments up with it out of the gel. When the fragments reach the
membrane, they stick to the membrane and remain there. Because the
wick, gel, membrane, and stack of paper lay directly on top of each other,
when the DNA fragments are transferred up onto the membrane, they
form the same pattern that they formed in the gel. After the transfer is
complete, the DNA-containing membrane is rinsed and the denatured DNA
molecules are fixed to it through heating or exposure to ultraviolet light.This membrane, with the fixed, single-stranded DNA, is now ready for
hybridization with the desired probe.
As a first step in the hybridization procedure, the membrane is immersed
in a prehybridization buffer that prevents the probe from binding to the
membrane in a nonspecific manner. After this prehybridization step, the
membrane is transferred to the hybridization solution containing the labeled
probe. The composition of the hybridization solution and the hybridization
conditions vary depending upon the probe used and the DNA sequence youwish to detect. After the hybridization is finished, the membrane is rinsed
repeatedly under conditions that will remove unhybridized and
nonspecifically bound probe, but that will not disrupt hydrogen bonds
between the probe and the target sequence in the sample DNA.
The final step in Southern hybridization is to detect the hybridized probe.
In this kit, the probe is attached to a molecule called biotin. To detect this
biotin-labeled probe, the hybridized membrane is soaked in a solution
containing a two-component molecule. One component is streptavidin, a
molecule that binds tightly to biotin. The other component is the enzyme,
alkaline phosphatase. The alkaline phosphatase protein and the attached
streptavidin together are called a conjugate, or a protein conjugate. During
the time that the membrane is soaked in the solution containing the
streptavidin-alkaline phosphatase conjugate, the streptavidin binds tightly
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to the biotinylated probe. The alkaline phosphatase becomes attached to
the probe by virtue of its bond to streptavidin. Once the incubation step to
bind the streptavidin-alkaline phosphatase conjugate to the biotin-labeled
probe is finished, the membrane is rinsed to remove the unbound conjugate.
Finally, the membrane is placed in a color development solution containing
two components: 5-bromo-4-chloro-3-indolyphosphate (BCIP) and nitro
blue tetrazolium (NBT). The alkaline phosphatase portion of the conjugate
removes a phosphate group from BCIP; the resulting product dimerizes to
form a dark blue precipitate. The dimerization reaction also releases
hydride ions that reduce the NBT; the reduced NBT forms a purple
precipitate. Since the alkaline phosphatase is bound to the probe via its
connection to streptavidin, the precipitates from its reaction with BCIP and
NBT form where the probe is bound to the membrane, thereby indicating
the location of the DNA fragments hybridized to the probe.
Student PreparationBefore attempting this exercise, students should be familiar with the theory
of restriction enzyme analysis and the mechanics of running gels. They
should have been introduced to the concepts of hybridization analysis, as well.
Further information on hybridization analysis as well as paper-and-pencil
exercises that illustrate the concepts can be found in Recombinant DNA
and Biotechnology: A Guide for Teachersby Kreuzer and Massey
(2001; ASM Press, Washington, DC; Carolina Biological Supply catalog
#RN-21-2218).
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MaterialsIncluded in the kit
Store these materials
at room temperature:
500 mL 2 denaturation buffer
1 L 2 neutralization buffer
1.5 L 20 SSC
250 mL prehybridization buffer
10 g blocking agent
500 mL Buffer 3
50 mL 10% SDS
12 staining trays (weigh boats)
12 pairs gloves
3 10- 15-cm positively charged nylon membranes
8 sheets Whatman 3MM filter paper
8 hybridization bags
100 mL bottle NBT/BCIP color development solution
6 Student Guides
Needed, but not supplied:
electrophoresis equipment for 6 groups
1.0% agarose gel, electrophoresis buffer, and stain for 6 gels
6 small, transparent metric rulers
6 pencils
6 scissors
stacks of brown paper towels
6 plastic containers with tight-fitting lids
6 shallow containers, approximately 28 51 cm, for transfer
6 400-mL beakers (1 per group)
6 flat pieces of plastic or glass, 8 10 cm or a little larger
Parafilm or plastic wrap
6 100-mL graduated cylinders
distilled or deionized water
water baths
gel photography equipment
access to oven (80C)
Store these materials at 4C
and keep on ice:
30 L biotinylated
oligonucleotide probe
100 L streptavidin-alkaline
phosphatase conjugate
6 tubes of each DNA
sample: /EcoRI, /HindIII
and /BstEII (may also be
stored frozen)
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SchedulingThere are several steps to Southern hybridization analysis. Letters
correspond to the steps of the laboratory activities in this kit.
A. Running, staining, and photographing the gel.
The time this step requires depends upon the staining system
used. The gel takes about 1 hr to run at 130 V and must be
photographed before treatment for the transfer. Ethidium bromide
is the fastest stain, requiring only 15 min. If CarolinaBLU is
incorporated into the gel (this does not interfere with hybridization
analysis), the gel can be stained for only 15 min and destained for
1530 min with continuous changes of water.
B. Treating the gel (denaturation) and setting up the transfer stack.
1 hr for treatment and 1015 min to assemble the stack
C., D. Overnight transfer.
E. Washing and baking the membrane.
30 min for washing and a minimum of 30 min for baking
F., G. Prehybridizing and hybridizing the membrane.
Prehybridize 90 min to overnight; hybridize overnightH. Washing the membrane and developing the color.
70 min for the washes and color development; 12 hr for bands
to develop
Suggested schedules
Day 1
Day 2
Day 3
Day 4
Day 5
(Step A) Run the gels. The instructor may need to destain and
photograph them. Leave the gels overnight.
(Steps B, C, D) Treat the gels (denaturation) for transfer and set
up the transfer stacks.
(Steps E, F) Wash and bake the membranes. Put the membranes
into prehybridization buffer and incubate them overnight.
(Step G) Put the membranes into hybridization buffer
(a 10-minute exercise). Leave them overnight.(Step H) Wash the membranes and perform the color
development reactions.
90-minute Classes (Experiment can be completed in 5 days.)
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7. Prehybridization Buffer and Hybridization Buffer: Hybridization Buffer
has the same composition as Prehybridization Buffer, except that
Hybridization Buffer has biotinylated oligonucleotide probe added.
Dissolve 2.5 g of blocking agent in 250 mL of Prehybridization Buffer.
Stir well. The solution will be white, but no particulate matter shouldremain. Remove 100 mL to a separate, clean container marked
Hybridization Buffer, 100 mL. Store both containers in the refrigerator
until use. Just before the hybridization (Step G), add 30 L of
biotinylated oligonucleotide probe to the 100 mL solution to make the
Hybridization Buffer.
8. Buffer 2: Dissolve 7.5 g of blocking agent in 750 mL of Buffer 1 (made in
Preparation Step 3). Stir well. The solution will become white, but no
solid particles should remain. Remove 100 mL of the Buffer 2 that you
just made to a clean container labeled 100 mL of Buffer 2 for SA-AP.
The 100 L streptavidin-alkaline-phosphatase conjugate will be added
to this 100 mL on the day of use (Step H7). Store both containers in the
refrigerator until use.
9. Buffer 3: Use as supplied.
10. Prepare membranes: While wearing gloves, cut each 10- 15-cm nylon
membrane into two 10- 7.5-cm membranes. Handle the membranes
by the corners and edges. Do not fold, rub, or crinkle them. Replace
them between their paper liners and put them back into the zipping bag.
Daily preparation
Before Step A, preparing and running a 1.0% agarose gel
Depending on your schedule, you may need to cast the gels in
advance so that students can immediately load their samples.
Each group will need:
1 1.0% agarose gel
1 sample each: cut w/EcoRI, cut w/HindIII, and cut w/BstEII DNA
equipment for loading the gel
The photography station should also have a small, transparent, metric
ruler. Students must lay the ruler on the edge of the gel so that, from
the photograph, they can tell the distance that each individual DNA
band has migrated away from the gel wells.
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Before Steps B, C, and D, transfer procedures
Each group will need:
1 plastic container with a tight-fitting lid
150 mL denaturation buffer
150 mL neutralization buffer
1 weigh boat
300 mL 10 SSC
1 7.5- 10-cm nylon membrane
1 sheet Whatman 3MM filter paper
1 pencil
1 pair of gloves
scissors
Parafilm or plastic wrap
brown paper towels
100-mL graduated cylinders
a means to measure 10 mL
1 400-mL beaker
1 flat piece of plastic or glass, 8 10 cm or slightly larger
Before Step E, taking down the stack, rinsing the membrane, and
baking the gel
Preheat oven to 7080C.
Each group will need:
1 plastic container with a tight-fitting lid
100 mL 2 SSC
2 approximately 11- 12-cm sheets of Whatman paper (cut 6 from
each of the two remaining pieces of Whatman 3MM paper)
Before Step F, prehybridization
Preheat water bath to 50C (42C is adequate, if yours does not get
that warm)
Each group will need:
10 mL Prehybridization Buffer
1 hybridization bag
1 pair of gloves (should be left from previous labs)
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Before Step G, hybridization
Prepare the Hybridization Buffer. Add 2030 L biotinylated probe
(contents of the tube) to the set-aside container labeled
Hybridization Buffer, 100 mL from Preparation Step 7.
Each group will need:
10 mL Hybridization Buffer
Before Step H, washing and probe detection (color development)
So that students can perform the third wash step at 50C, pre-warm
600 mL of Wash Buffer to 50C (42C is adequate, if this is as warm as
your bath gets). Pre-warm the buffer by placing a closed container
holding 600 mL of buffer into a water bath. A tightly closed container
of Wash Buffer can be left in a water bath overnight.
Students can perform the third wash step by floating the containers,
with the lids on, holding the membranes and pre-warmed buffer in the
water bath during the wash. Another way to set this up is to fill a few
insulated foam containers with 50C water just prior to use. The hot
taps of many sinks provide water that is close to 50C.
Prepare streptavidin-alkaline phosphatase (SA-AP) conjugate solutionjust before use. On the day you are to use it, add 100 L (contents of
the tube) of SA-AP conjugate to the 100 mL of Buffer 2 set aside and
labeled 100 mL of Buffer 2 For SA-APfrom Preparation Step 8.
Each group will need:
1 100-mL graduated cylinder for measuring buffer volumes
200 mL wash buffer at room temperature
100 mL wash buffer at water bath temperature (4250C)
300 mL Buffer 1
100 mL Buffer 2 (can be put at room temperature for the lab period)
1015 mL SA-AP solution
50 mL Buffer 3
15 mL NBT/BCIP color development solution
(solution comes ready-to-use)
a clean weigh boatgloves
Note: To save you money, we have not included much excess material
in this kit. It is important that students use the recommended amounts
of solutions.
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Laboratory Procedures
A. Prepare and run a 1.0% agarose gel
1. Each student group should have (or prepare) a 1.0% agarose gel.An 8- 10-cm gel requires 50 mL of agarose solution. For 1.0%
agarose, use 0.5 g of agarose per 50 mL of 1 TBE buffer.
(CarolinaBLU Gel and Buffer Stain may be incorporated into the gels
and the TBE electrophoresis buffer to decrease the staining time.
Follow the instructions included with the stain.)
2. Noting the order, load the three DNA samples in adjacent lanes; if
possible, leave an outside lane empty so that the gel can be trimmed
if it is more than 7.5 cm wide. The entire contents of each sampletube should be loaded in a well. (This amount of DNA will look
overloaded if stained with ethidium bromide, but works well for
the transfer and detection.)
3. Run the bromophenol blue dye to the bottom of the gel. This takes
about 1 hr at 130 V.
4. Use CarolinaBLU or other stain of your choice to visualize the DNA.
The gel must be photographed alongside a ruler before treatment fortransfer. Lay a transparent ruler on the gel so that you can determine
the distance from the wells to a particular DNA band from the
photograph (Figure 1).
5. Gels can be stored refrigerated overnight in 1 TBE buffer beforetransfer, if necessary. Refrigerating the gels during any overnight
storage will help prevent the DNA bands in the gel from diffusing.
1
2
3
4
5
6
7
8
9
10
1
2
3
4
Figure 1.
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B.Treat the gels and set up the transfer stacks
Note: The treatment involves four 15-min incubations. During theseincubations, prepare the materials for the transfer stack.
1. Place the gel in a small plastic container, add 75 mL of 1 DenaturationBuffer, and incubate for 15 min at room temperature. Occasionally,
gently agitate the container.
2. Pour off the Denaturation Buffer, add 75 mL of fresh 1 Denaturation
Buffer, and incubate for 15 min, as before.
3. Pour off the Denaturation Buffer, add 75 mL of 1 Neutralization Buffer,
and incubate for 15 min at room temperature. Occasionally, gently
agitate the container.
4. Pour off the Neutralization Buffer, add 75 mL of fresh 1 Neutralization
Buffer, and incubate for 15 min, as before. Leave the gel in the
Neutralization Buffer until you assemble the transfer stack (Step C).
C. Prepare materials for the transfer stack
1. Prepare the membrane.
Note: The positively charged nylon membrane is vulnerable to abrasion
and grease. Wear gloves, and handle it by the corners at all times. Do not
bend or abrade it. Use a pencil to write DNA and your groups initials insmall letters at the center of the 7.5-cm side of the 7.5- 10-cm membrane.
(The side you write on will contact the gel.) Put a small amount of
deionized or distilled water in your weigh boat and float the membrane
on the water until it is thoroughly wet. Then, remove the membrane
from the water, replace the water with a small amount of 10 SSC, and
float the membrane on the solution. Leave it there until you assemble
the transfer stack.
2. Prepare the Whatman3MM filter paper.
Cut two 9- 18-cm rectangles of
Whatman 3MM paper to serve as
wicks. Cut two 7.5- 10-cm
rectangles for the transfer stack
(see diagram at right).
3. Prepare the paper towels.
Cut enough 7.5- 10-cm
rectangles of brown paper
towels to form a 2-inch stack
when compressed.
13
9 cm 9 cm
10 cm
10 cm
28.5 cm
23cm
18 cm 18 cm7.5 cm
7.5 cm
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D. Set up the transfer stack (see Figure 2)
Note: Always wear gloves when handling the membrane and wicks;
otherwise, oil and grease from your fingers will interfere with the transfer
and subsequent hybridization.
1. Place 250 mL of 10 SSC in a shallow container measuring
approximately 11 20 cm.
2. Wet the two paper wicks in the 10 SSC. Leave them in the 10 SSC
until you perform Step 4.
3. Place an inverted gel casting tray or other support (see Figure 2) in the
shallow dish. The 10 SSC should not cover the support.
4. Lay the two wicks (one on top of the other) over the inverted casting
tray, so that the ends of both wicks are well submerged in the 10 SSC.
5. Make sure that no air bubbles are trapped between the wicks.
Rolling over them with a pencil or plastic pipet can help squeeze
out air bubbles.
6. Remove the gel from the neutralization buffer. Lay it, with the open
side of the wells facing down, on the wicks (on top of the inverted
casting tray). Cut off a small piece of one lower corner of the gel and
record which corner you cut in relation to the position of the DNA on
the gel. Make sure no air bubbles are trapped between the gel and the
wicks. Gently roll a pencil or pipet over the gel to eliminate bubbles.
7. Remove the membrane from the 10 SSC in the weigh boat and
carefully lay it on the gel, with the side you wrote on contacting the
gel. The membrane is narrower than an 8- 10-cm gel, so be sure that
the lanes of the gel that contain DNA are covered. Trim off the
exposed portion of the gel, carefully avoiding the wick.
400 ml
Paper towels
Nitrocellulose filteror nylon membrane
Whatman 3MM
paper wicks
Transfer buffer
Weight (500 g)
Glass plate
Whatman 3MM paper
Gel
Support(inverted casting tray)
Plastic wrap
Figure 2. Transfer stack
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8. Cut off a small lower corner of the membrane to match the cut lower
corner of the gel. (This will help you orient the membrane properly
after hybridization.)
9. Lay strips of plastic wrap or Parafilm
around the gel so that the rest ofthe wick area on the casting tray is covered, if it is not already. If your
gel has areas not covered by the membrane, cover them too, but do
not cover the membrane. This ensures that the transfer buffers only
migration path is through the gel and membrane (i.e., it prevents the
edge of a paper towel or other stack components from accidentally
contacting the wick or uncovered portions of the gel).
10. Place the two dry 7.5- 10-cm rectangles of Whatman 3MM paper
neatly on top of the membrane.
11. Place the stack of dry, cut paper towels neatly on top of the Whatman
3MM paper.
12. On top of the paper towels, place a flat piece of plastic or glass. On top
of this, for weight (~500 g), place a 400-mL beaker full of water.
13. Allow the transfer stack to sit overnight.
E. Take down the stack, rinse the membrane, and bake the gelThe lower part of the paper towel stack should be completely saturated
with buffer, but the paper towels themselves should not be in contact with
the wicks or with the buffer in the reservoir. There should still be buffer in
the reservoir. The wick ends should still be submerged.
1. Place 100 mL of 2 SSC into a
plastic container with a tight-
fitting lid. You will use this inStep 5.
2. Remove the 400-mL beaker
weight from the transfer stack.
Discard the paper towels to
expose the Whatman 3MM
paper sheets.
3. With gloved hands, carefully
leaving what remains of the
stack together as a unit, turn
the gel, membrane, and
Whatman 3MM paper over.
With a soft lead pencil, pierce the wells of the gel to mark the locations
of the wells on the membrane (Figure 3).
Peel off gel
Figure 3. Use a pencil to pierce the wellsand mark their locations on the membrane.
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4. Peel the gel off the membrane and discard it. If you used
CarolinaBLU or methylene blue to stain the gel, the
high-molecular-weight DNA bands will still be visible: this is
normal, and does not mean that transfer failed to occur.
5. Peel the nylon membrane away from the two sheets of 3MM paper
and place it in the 2 SCC prepared in Step 1. Gently agitate the
container for 30 min. Note: The membrane should not bend or crinkle.
Do not reduce the wash time.
6. With gloved hands or with blunt forceps, place the nylon membrane
on a piece of Whatman 3MM filter paper. Allow it to air dry for at least
5 min. Then, write your group name on a second piece of Whatman
paper, place it over the membrane, and then tape the pieces ofWhatman paper together, avoiding the membrane.
7. Bake this filter paper and membrane sandwich for 3060 min in
a 7080C oven.
8. The membrane may be stored indefinitely at room temperature.
F. Prehybridization
1. With gloved hands or with blunt forceps, place the membrane all theway down into one of the hybridization bags provided. Handle the
membrane gently and only by the corners.
2. Pour or pipet 10 mL of Prehybridization Buffer into the bag. Starting at
the bottom, squeeze the bag gently to push most of the bubbles
toward the top. The goal is to have a thin layer of fluid covering the
membrane; air bubbles can prevent contact between the membrane
and the fluid. When most of the bubbles have been squeezed past the
zip-lock strip (expect some buffer to be lost), carefully seal the bag.
Make certain that the bag is sealed.
3. Tape the top of the bag to the inside wall of a 50C water bath,
allowing the membrane-containing portion of the bag to hang in the
water. If there are still bubbles in the bag, move them to the area of
the bag above the membrane.
4. Incubate 90 min to overnight.
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G. Hybridization
1. Remove the bag from the water bath, open the bag, and pour out the
Prehybridization Buffer. Immediately add 10 mL of Hybridization Buffer.
(Hybridization Buffer has the same composition as PrehybridizationBuffer, except that the biotinylated oligonucleotide probe has been
added.) Do not allow the membrane to dry out at all between when the
Prehybridization Buffer is removed and the Hybridization Buffer is
added. Make sure that the probe has been added to the Hybridization
Buffer beforeyou pour off the Prehybridization Buffer.
2. Work bubbles out of the bag, as before.
3. Place the bag in the water bath, as before.
4. Hybridize overnight.
H. Washing and probe detection (color development)
Note: Follow these directions carefully. Do not reduce the Wash Buffer
volumes. Do not allow the membrane to dry out between buffer changes.
1. Place 100 mL of Wash Buffer into a plastic container with a tight-fitting lid.
2. Remove the nylon membrane from the hybridization bag and
immediately place it in the Wash Buffer in the plastic container.
Discard the bag and Hybridization Buffer. Agitate the membrane very
gently for 5 min at room temperature (it should not bend or tumble).
3. Pour off the Wash Buffer, immediately add 100 mL of fresh Wash
Buffer, and agitate gently for 5 min.
4. Pour off the Wash Buffer, add 100 mL 50C Wash Buffer, and agitate
gently for 5 min. If possible, float the container (with the lid on) in a
50C water bath during this wash step, to maintain temperature.
5. Pour off the Wash Buffer, immediately add 100 mL of Buffer 1 to the
container, and agitate gently for 5 min at room temperature.
6. Pour off Buffer 1, add 100 mL of Buffer 2, and agitate gently for 30 min
at room temperature. Do not reduce the agitation time.
7. Pour off Buffer 2, place the membrane (with the side labeled DNA
facing up) in a clean weigh boat, and immediately add 1015 mL of
streptavidin-alkaline phosphatase conjugate in Buffer 2. Then, rock themembrane very slowly at room temperature for 1015 min. The
solution should move quite slowly across the membrane.
8. Place the membrane back into the plastic container, immediately add
100 mL of Buffer 1, and agitate the container gently for 15 min at room
temperature (it should not bend or tumble). Rinse and dry the
weigh boat.
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9. Pour off Buffer 1, immediately add 100 mL of fresh Buffer 1, and agitate
the container gently at room temperature for 15 min, as before.
10. Remove the membrane and place it (with the side labeled DNA facing
up) in the clean weigh boat. Immediately add 50 mL of Buffer 3 and letit stand for 5 min at room temperature.
11. Pour off Buffer 3, immediately add 15 mL of fresh NBT/BCIP color
development solution, and place the weigh boat in a dark place such
as a cabinet or drawer. Color development will require 30 min to 2 hr;
check the membrane periodically, but do not move it. When the bands
have appeared and darkened, pour off the color development solution
and replace it with distilled or deionized water. Let the membrane stand
in water for 5 to 30 min, then remove the membrane and allow it to airdry. Store the membrane in a dark place, as light will fade the bands.
Analysis of ResultsThe oligonucleotide probe is a 21-base sequence from the bacteriophage
lambda genome. The data from this exercise allows students to determine
the approximate location of that sequence within the lambda genome.
First, students need restriction maps of lambda for the three enzymes,HindIII, EcoRI, and BstEII. The restriction site locations for these enzymes
are listed below. Have students draw maps from this data and determine
the fragment lengths that would result from digestion of lambda DNA with
these enzymes.
Next, students must determine which bands from each of the digests
hybridized to the probe. To do this, they must measure the distance
between the marks on their membrane (showing the locations of the wells)
down to the band that developed after hybridization. Use this measurement
and similar measurements made using the photograph of the gel with the
ruler to determine which bands on the gel correspond to the bands that
develop on the membrane after hybridization. Use the ruler in the
photograph to measure the gel in the photograph. There can be some error
in this process because the pencil marks indicating the well location may
not line up exactly with the location of the wells. In addition, the transfer
may not have been exactly vertical if the transfer stack was off balance.
The uncertainty created by this potential error can be dealt with through
the following reasoning. The oligonucleotide probe is only one 21-base
sequence. In each digest, it will hybridize only to the band that contains
that sequence, so the bands that hybridize to the probe must contain an
area of overlap. If students have to decide which of two bands actually
hybridized to the probe, they can compare the bands locations in the lambda
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genome to the locations of the hybridizing bands from the other digests.
Remember that the hybridizing bands in each of the digests must contain at
least one small common area. This area is the location of the probe sequence
in the lambda genome.
Restriction site locations within the lambda chromosome
(48,502 base pairs)
EcoRI sites
21,226 26,104 31,747 39,168 44,972
HindIII sites
23,130 25,157 27,479 36,895 37,459 37,584 44,141
BstEII sites
5687 7058 8322 9024 13,348 13,572 13,689
16,012 17,941 25,183 30,005 36,374 40,049
The probe sequence stretches from base 39,778 to 39,799.
Further ReadingBloom, M., G. Freyer, and D. Micklos. 1995. Laboratory DNA Science.
Benjamin/Cummings, Menlo Park, CA.
Kreuzer, H., and A. Massey. 2001. Recombinant DNA and Biotechnology:
A Guide For Teachers. American Society for Microbiology Press,
Washington, DC. (Carolina Biological Supply catalog #RN-21-2218)
Micklos, D., and G. Freyer, with David A. Crotty. 2003. DNA Science: A First
Course, 2nd Edition. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY. (Carolina Biological Supply catalog #RN-21-2209)
Mullis, K. 1990. The unusual origin of the polymerase chain reaction.
Scientific American 262(4):5665.
Nakamura, Y., M. Carlson, K. Krapco, and R. White. 1988. Isolation and
mapping of a polymorphic DNA sequence (pMCT118) on
chromosome 1p. Nucleic Acids Research 16:9364.
National Research Council. 1996. The Evaluation of Forensic DNA Evidence.
National Academy Press, Washington, DC.
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Student Guide Name
21-1215 Date
Southern Hybridization Experiment Kit
1 2 0 0 4 C a r o l i n a B i o l o g i c a l S u p p l y C o m p a n y
IntroductionDuring many procedures, including DNA library screening, DNA-based disease diagnosis, and DNA
fingerprinting, it is important to know whether a specific DNA sequence is present in a DNA sample and
where it is located with respect to restriction enzyme sites. Restriction enzyme digestion, electrophoresis, and
staining allow us to cut DNA molecules into reproducible pieces and to determine the size of these pieces.
However, restriction enzyme analysis alone does not provide information about DNA sequences present
within the fragments. Southern hybridization analysis combines restriction enzyme analysis and
hybridization analysis to provide this kind of information. Combining these techniques reveals which
fragments from a restriction digest (if any) contain a specific DNA sequence.
In brief, hybridization analysis involves separating (denaturing) the strands of the DNA molecules to be
analyzed and then mixing those separated strands with many copies of a single-stranded DNA or RNA
molecule, called a probe. The probe contains a sequence complementary to the nucleotide sequence of
interest. When a probe is mixed with single-stranded (denatured) DNA under the right conditions, hydrogen
bonds form between the probe and its complementary sequence in the DNA sample being analyzed. The
formation of hydrogen bonds between two complementary strands to create a double-stranded complex is
called hybridization, or annealing. When a DNA or RNA probe bonds to its complementary sequence in the
DNA being analyzed, the probe is said to be annealing (or hybridizing) to the sample DNA. Hybridization
analysis can be performed either in solution or with one component attached to some kind of solid support.
For Southern hybridization analysis, the sample DNA is attached to a solid support, such as a nitrocellulose
or nylon membrane.
Hybridization involves several steps. First, the probe and sample DNA are allowed to hybridize under
the appropriate conditions. The correct temperature, incubation times, and buffer conditions must
be used. Next, the sample DNA is washed using conditions that will remove unhybridized probe but not
the hybridized probe. Finally, the sample DNA is tested for the presence of the hybridized probe. The
probe is labeled with a radioactive molecular tag (or some other tag) that allows it to be detected
following hybridization.
To begin Southern hybridization analysis, the sample DNA is digested with restriction enzymes and the
resulting fragments are separated by agarose gel electrophoresis. The DNA must then be transferred from
the agarose gel to a solid support prior to hybridization. In 1975, a scientist named Southern published a
method for transferring DNA fragments from an agarose gel to a membrane in a manner that preserved the
arrangement of the fragments as they existed in the gel. Because of the scientists name, this transfer
method is known as Southern transfer or Southern blotting.
To perform Southern transfer, the agarose gel is first soaked in a basic solution to denature the DNA
fragments. After an additional soaking step to neutralize the base, the gel is placed on a long piece of blotting
paper with the ends of the paper suspended in a reservoir of salt solution. A nitrocellulose or nylon
membrane is then laid directly on top of the gel. Blotting paper and a stack of dry absorbent paper (such as
paper towels) are then placed on the membrane (see Fig. 2, page 3). The blotting paper acts like a wick.
Driven by capillary action, fluid is drawn from the reservoir up though the gel and into the stack of dry paper.
As the fluid migrates up through the gel, it carries the denatured DNA fragments up with it out of the gel.
When the fragments reach the membrane, they stick to the membrane and remain there. Because the wick,
gel, membrane, and stack of paper lay directly on top of each other, when the DNA fragments are
transferred up onto the membrane, they form the same pattern that they formed in the gel. After the
transfer is complete, the DNA-containing membrane is rinsed and the denatured DNA molecules are fixedto it through heating or exposure to ultraviolet light. This membrane, with the fixed, single-stranded DNA, is
now ready for hybridization with the desired probe.
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As a first step in the hybridization procedure, the membrane is immersed in a prehybridization buffer that
prevents the probe from binding to the membrane in a nonspecific manner. After this prehybridization step,
the membrane is transferred to the hybridization solution containing the labeled probe. The composition of
the hybridization solution and the hybridization conditions vary depending upon the probe used and the DNA
sequence you wish to detect. After the hybridization is finished, the membrane is rinsed repeatedly under
conditions that will remove unhybridized and nonspecifically bound probe, but that will not disrupt
hydrogen bonds between the probe and the target sequence in the sample DNA.
The final step in Southern hybridization is to detect the hybridized probe. In this kit, the probe is attached to
a molecule called biotin. To detect this biotin-labeled probe, the hybridized membrane is soaked in a
solution containing a two-component molecule. One component is streptavidin, a molecule that binds
tightly to biotin. The other component is the enzyme, alkaline phosphatase. The alkaline phosphatase
protein and the attached streptavidin together are called a conjugate, or a protein conjugate. During the
time that the membrane is soaked in the solution containing the streptavidin-alkaline phosphatase
conjugate, the streptavidin binds tightly to the biotinylated probe. The alkaline phosphatase becomes
attached to the probe by virtue of its bond to streptavidin. Once the incubation step to bind thestreptavidin-alkaline phosphatase conjugate to the biotin-labeled probe is finished, the membrane is
rinsed to remove the unbound conjugate.
Finally, the membrane is placed in a color development solution containing two components: 5-bromo-4-
chloro-3-indolyphosphate (BCIP) and nitro blue tetrazolium (NBT). The alkaline phosphatase portion of the
conjugate removes a phosphate group from BCIP; the resulting product dimerizes to form a dark blue
precipitate. The dimerization reaction also releases hydride ions that reduce the NBT; the reduced NBT
forms a purple precipitate. Since the alkaline phosphatase is bound to the probe via its connection to
streptavidin, the precipitates from its reaction with BCIP and NBT form where the probe is bound to the
membrane, thereby indicating the location of the DNA fragments hybridized to the probe.
Laboratory ProceduresNote: Southern hybridization involves many steps. To ensure success, carefully follow these instructions for
the many steps of Southern hybridization. Do not hurry; measure all volumes carefully and perform
incubations exactly as described. Be especially careful when handling the membrane.
A. Prepare and run a 1.0% agarose gel
1. Each student group should have (or prepare) a 1.0% agarose gel.
An 8- 10-cm gel requires 50 mL of agarose solution. For 1.0% agarose, use
0.5 g of agarose per 50 mL of 1 TBE buffer. (CarolinaBLU Gel and Buffer
Stain may be incorporated into the gels and the TBE electrophoresis buffer to
decrease the staining time. Follow the instructions included with the stain.)
2. Noting the order, load the three DNA samples in adjacent lanes; if possible,
leave an outside lane empty so that the gel can be trimmed if it is more than
7.5 cm wide. The entire contents of each sample tube should be loaded in a
well. (This amount of DNA will look overloaded if stained with ethidium
bromide, but works well for the transfer and detection.)
3. Run the bromophenol blue dye to the bottom of the gel. This takes about 1 hr
at 130 V.
4. Use CarolinaBLU or other stain of your choice to visualize the DNA. The gel
must be photographed alongside a ruler before treatment for transfer. Lay a
transparent ruler on the gel so that you can determine the distance from the
wells to a particular DNA band from the photograph (Figure 1).
5. Gels can be stored refrigerated overnight in 1 TBE buffer before transfer, if necessary. Refrigerating
the gels during any overnight storage will help prevent the DNA bands in the gel from diffusing.
1
2
3
4
5
6
7
8
9
10
1
2
3
4
Figure 1.
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B. Treat the gels and set up transfer stacks
Note: The treatment involves four 15-min incubations. During these incubations, prepare the materials forthe transfer stack.
1. Place the gel in a small plastic container, add 75 mL of 1 Denaturation Buffer, and incubate for 15 min
at room temperature. Occasionally, gently agitate the container.
2. Pour off the Denaturation Buffer, add 75 mL of fresh 1 Denaturation Buffer, and incubate for 15 min,
as before.
3. Pour off the Denaturation Buffer, add 75 mL of 1 Neutralization Buffer, and incubate for 15 min at room
temperature. Occasionally, gently agitate the container.
4. Pour off the Neutralization Buffer, add 75 mL of fresh 1 Neutralization Buffer, and incubate for 15 min,
as before. Leave the gel in the Neutralization Buffer until you assemble the transfer stack (Step C).
C. Prepare materials for the transfer stack
1. Prepare the membrane.
Note: The positively charged nylon membrane is vulnerable to abrasion and grease. Wear gloves, and
handle it by the corners at all times. Do not bend or abrade it. Use a pencil to write DNA and your
groups initials in small letters at the center of the 7.5-cm side of the 7.5- 10-cm membrane. (The side
you write on will contact the gel.) Put a small amount of deionized or distilled water in your weigh boat
and float the membrane on the water until it is thoroughly wet. Then, remove the membrane from the
water, replace the water with a small amount of 10 SSC, and float the membrane on the solution.
Leave it there until you assemble the transfer stack.
2. Prepare the Whatman 3MM filter paper.
Cut two 9- 18-cm rectangles of Whatman 3MM paper to
serve as wicks. Cut two 7.5- 10-cm rectangles for the
transfer stack (see diagram at right).
3. Prepare the paper towels.
Cut enough 7.5- 10-cm rectangles of brown paper towels
to form a 2-inch stack when compressed.
D. Set up the transfer stack (see Figure 2)
Note: Always wear gloves when handling the membrane and
wicks; otherwise, oil and grease from your fingers will
interfere with the transfer and subsequent hybridization.
1. Place 250 mL of 10 SSC in a
shallow container measuring
approximately 11 20 cm.
2. Wet the two paper wicks in the
10 SSC. Leave them in the
10 SSC until you perform Step 4.
3. Place an inverted gel casting tray
or other support (see Figure 2) in
the shallow dish. The 10 SSC
should not cover the support.
4. Lay the two wicks (one on top of the other) over the inverted casting tray, so that the ends of both wicks
are well submerged in the 10 SSC.
9 cm 9 cm
10 cm
10 cm
28.5 cm
23cm
18 cm 18 cm7.5 cm
7.5 cm
400 ml
Paper towels
Nitrocellulose filteror nylon membrane
Whatman 3MMpaper wicks
Transfer buffer
Weight (500 g)
Glass plate
Whatman 3MM paper
Gel
Support(inverted casting tray)
Plastic wrap
Figure 2. Transfer stack
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4 2 0 0 4 C a r o l i n a B i o l o g i c a l S u p p l y C o m p a n y
5. Make sure that no air bubbles are trapped between the wicks. Rolling over them with a pencil or
plastic pipet can help squeeze out air bubbles.
6. Remove the gel from the neutralization buffer. Lay it, with the open side of the wells facing down, on
the wicks (on top of the inverted casting tray). Cut off a small piece of one lower corner of the gel and
record which corner you cut in relation to the position of the DNA on the gel. Make sure no air bubbles
are trapped between the gel and the wicks. Gently roll a pencil or pipet over the gel to eliminate bubbles.
7. Remove the membrane from the 10 SSC in the weigh boat and carefully lay it on the gel, with the side
you wrote on contacting the gel. The membrane is narrower than an 8- 10-cm gel, so be sure that the
lanes of the gel that contain DNA are covered. Trim off the exposed portion of the gel, carefully
avoiding the wick.
8. Cut off a small lower corner of the membrane to match the cut lower corner of the gel. (This will help
you orient the membrane properly after hybridization.)
9. Lay strips of plastic wrap or Parafilm
around the gel so that the rest of the wick area on the castingtray is covered, if it is not already. If your gel has areas not covered by the membrane, cover them too,
but do not cover the membrane. This ensures that the transfer buffers only migration path is through
the gel and membrane (i.e., it prevents the edge of a paper towel or other stack components from
accidentally contacting the wick or uncovered portions of the gel).
10. Place the two dry 7.5- 10-cm rectangles of Whatman 3MM paper neatly on top of the membrane.
11. Place the stack of dry, cut paper towels neatly on top of the Whatman 3MM paper.
12. On top of the paper towels, place a flat piece of plastic or glass. On top of this, for weight (~500 g),
place a 400-mL beaker full of water.
13. Allow the transfer stack to sit overnight.
E. Take down the stack, rinse the membrane, and bake the gelThe lower part of the paper towel stack should be completely saturated with buffer, but the paper towels
themselves should not be in contact with the wicks or with the buffer in the reservoir. There should still be
buffer in the reservoir. The wick ends should still be submerged.
1. Place 100 mL of 2 SSC into a plastic container with a tight-fitting
lid. You will use this in Step 5.
2. Remove the 400-mL beaker weight from the transfer stack. Discard
the paper towels to expose the Whatman 3MM paper sheets.
3. With gloved hands, carefully leaving what remains of the stack
together as a unit, turn the gel, membrane, and 3MM paper over.
With a soft lead pencil, pierce the wells of the gel to mark thelocations of the wells on the membrane (Figure 3).
4. Peel the gel off the membrane and discard it. If you used
CarolinaBLU or methylene blue to stain the gel, the
high-molecular-weight DNA bands will still be visible; this is
normal and does not mean that transfer failed to occur.
5. Peel the nylon membrane away from the two sheets of 3MM
paper and place it in the 2 SSC prepared in Step 1. Gently
agitate the container for 30 min. Note: The membrane should not
bend or crinkle. Do not reduce the wash time.
6. With gloved hands or with blunt forceps, place the nylon membrane on a piece of Whatman 3MM filter paper.
Allow it to air dry for at least 5 min. Then, write your group name on a second piece of Whatman paper, placeit over the membrane, and then tape the pieces of Whatman paper together, avoiding the membrane.
7. Bake this filter paper and membrane sandwich for 3060 min in a 7080C oven.
8. The membrane may be stored indefinitely at room temperature.
Peel off gel
Figure 3. Use a pencil to pierce
the wells and mark their
locations on the membrane.
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F. Prehybridization
1. With gloved hands or with blunt forceps, place the membrane all the way down into one of thehybridization bags provided. Handle the membrane gently and only by the corners.
2. Pour or pipet 10 mL of Prehybridization Buffer into the bag. Starting at the bottom, squeeze the bag
gently to push most of the bubbles toward the top. The goal is to have a thin layer of fluid covering the
membrane; air bubbles can prevent contact between the membrane and the fluid. When most of the
bubbles have been squeezed past the zip-lock strip (expect some buffer to be lost), carefully seal the
bag. Make certain that the bag is sealed.
3. Tape the top of the bag to the inside wall of a 50C water bath, allowing the membrane-containing
portion of the bag to hang in the water. If there are still bubbles in the bag, move them to the area of
the bag above the membrane.
4. Incubate 90 min to overnight.
G. Hybridization
1. Remove the bag from the water bath, open the bag, and pour out the Prehybridization Buffer.
Immediately add 10 mL of Hybridization Buffer. (Hybridization Buffer has the same composition as
Prehybridization Buffer, except that the biotinylated oligonucleotide probe has been added.) Do not allow
the membrane to dry at all between when the Prehybridization Buffer is removed and the Hybridization
Buffer is added. Make sure that the probe has been added to the Hybridization Buffer beforeyou pour off
the Prehybridization Buffer.
2. Work bubbles out of the bag, as before.
3. Place the bag in the water bath, as before.
4. Hybridize overnight.
H. Washing and probe detection (color development)
Note: Follow these directions carefully. Do not reduce the Wash Buffer volumes. Do not allow the
membrane to dry out between buffer changes.
1. Place 100 mL of Wash Buffer into a plastic container with a tight-fitting lid.
2. Remove the nylon membrane from the hybridization bag and immediately place it in the Wash Buffer in
the plastic container. Discard the bag and Hybridization Buffer. Agitate the membrane very gently for 5
min at room temperature (it should not bend or tumble).
3. Pour off the Wash Buffer, immediately add 100 mL of fresh Wash Buffer, and agitate gently for 5 min.
4. Pour off the Wash Buffer, immediately add 100 mL of 50C Wash Buffer, and agitate gently for 5 min.
If possible, float the container (with the lid on) in a 50C water bath during this wash step, to
maintain temperature.
5. Pour off the Wash Buffer, immediately add 100 mL of Buffer 1 to the container, and agitate gently for
5 min at room temperature.
6. Pour off Buffer 1, add 100 mL of Buffer 2, and agitate gently for 30 min at room temperature. Do not
reduce the agitation time.
7. Pour off Buffer 2, place the membrane (with the side labeled DNA facing up) in a clean weigh boat, and
immediately add 1015 mL of streptavidin-alkaline phosphatase conjugate in Buffer 2. Then, rock the
membrane very slowly at room temperature for 1015 min. The solution should move quite slowly
across the membrane.
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8. Place the membrane back into the plastic container, immediately add 100 mL of Buffer 1, and agitate
the container gently for 15 min at room temperature (it should not bend or tumble). Rinse and dry the
weigh boat.
9. Pour off Buffer 1, immediately add 100 mL of fresh Buffer 1, and agitate the container gently at room
temperature for 15 min, as before.
10. Remove the membrane and place it (with the side labeled DNA facing up) in the clean weigh boat.
Immediately add 50 mL of Buffer 3 and let it stand for 5 min at room temperature.
11. Pour off Buffer 3, immediately add 15 mL of fresh NBT/BCIP color development solution, and place the
weigh boat in a dark place such as a cabinet or drawer. Color development will require 30 min to 2 hr;
check the membrane periodically, but do not move it. When the bands have appeared and darkened,
pour off the color development solution and replace it with distilled or deionized water. Let the
membrane stand in water for 5 to 30 min, then remove the membrane and allow it to air dry. Store the
membrane in a dark place, as light will fade the bands.
Analysis of Results
The oligonucleotide probe is a 21-base sequence from the bacteriophage lambda genome. The data from
this exercise allows you to determine the approximate location of that sequence within the lambda genome.
The restriction site locations for the enzymes HindIII, EcoRI and BstEII are listed below. Draw maps from this
data and determine the fragment lengths that would result from digestion of lamda DNA with these enzymes.
Determine which bands from each of the digests hybridized to the probe. To do this, measure the distance
between the marks on your membrane (showing the locations of the wells) down to the band that
developed after hybridization. Use this measurement and similar measurements made using the
photograph of the gel with the ruler to determine which bands on the gel correspond to the bands that
develop on the membrane after hybridization. Use the ruler in the photograph to measure the gel in thephotograph. There can be some error in this process because the pencil marks indicating the well location
may not line up exactly with the location of the wells. In addition, the transfer may not have been exactly
vertical if the transfer stack was off balance.
The uncertainty created by this potential error can be dealt with through the following reasoning. The
oligonucleotide probe is only one 21-base sequence. In each digest, it will hybridize only to the band that
contains that sequence, so the bands that hybridize to the probe must contain an area of overlap. If you have
to decide which of two bands actually hybridized to the probe, you can compare the bands locations in the
lambda genome to the locations of the hybridizing bands from the other digests. Remember that the
hybridizing bands in each of the digests must contain at least one small common area. This area is the
location of the probe sequence in the lambda genome.
Restriction site locations within the lambda chromosome(48,502 base pairs)
EcoRI sites
21,226 26,104 31,747 39,168 44,972
HindIII sites
23,130 25,157 27,479 36,895 37,459 37,584 44,141
BstEII sites
5687 7058 8322 9024 13,348 13,572 13,689 16,012 17,941 25,183
30,005 36,374 40,049
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