21-1215

Southern Hybridization Experiment Kit

Teachers Manual

400 ml

Weight (500 g)

Paper towels Nitrocellulose filter or nylon membrane Whatman 3MM paper wicks Transfer buffer Support (inverted casting tray)

Glass plate

Whatman 3MM paper Gel Plastic wrap

World-Class Support for Science & Math

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 this manual to plan your laboratory sessions. Some of the introduction to this kit was excerpted with permission from Recombinant DNA and Biotechnology: A Guide for Teachers by Kreuzer and Massey (2001; ASM Press, Washington, DC; Carolina Biological Supply catalog #RN-21-2218).

2004 Carolina Biological Supply Company

Printed in USA

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 singlestranded (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 3

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 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 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 4

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 Preparation
Before 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 Teachers by Kreuzer and Massey (2001; ASM Press, Washington, DC; Carolina Biological Supply catalog #RN-21-2218).

Materials
Included 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

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)

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) 6

Scheduling
There 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 overnight H. Washing the membrane and developing the color. 70 min for the washes and color development; 12 hr for bands to develop

Suggested schedules
90-minute Classes (Experiment can be completed in 5 days.)
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.

3-hour Laboratory Periods (Experiment requires three labs.)

(Steps A, B, C, D) Run, stain and photograph the gels. Treat the gels (denaturation) and set up the transfer stacks. (It may be necessary to prepare the gels before the lab so that they can be loaded immediately.) Transfer stacks should be taken down the next day. Membranes can be rinsed briefly in 2 SSC and left at room temperature until the next lab. (Steps E, F, G) Wash the membranes for 30 minutes in 2 SSC and bake them for 30 minutes. Prehybridize for 90 minutes, then add the hybridization solution. Leave the membranes until the next lab. (Step H) Wash the membranes and perform the color development reactions.

Day 1

Day 2

Day 3

Preparation
Southern hybridization analysis and nonradioactive detection require many different solutions. We have supplied concentrated stock solutions for preparing them; please follow the instructions carefully. It may take several hours to prepare all the solutions. Note: To save you money, we have not included much excess solution of any kind. It is important that students use the recommended amounts of solutions. After preparing the Prehybridization Buffer, Hybridization Buffer, and Buffer 2, store them in the refrigerator. Store other solutions at room temperature unless it is otherwise stated.

General
1. Denaturation Buffer: Dilute 500 mL of 2 denaturation buffer to 1 L of 1 with distilled or deionized water. 2. Neutralization Buffer: Dilute 500 mL of 2 neutralization buffer to 1 L of 1 with distilled or deionized water. 3. Buffer 1: Dilute 150 mL of 2 neutralization buffer with 2850 mL distilled or deionized water to make 3 L of Buffer 1. 4. 10 SSC: Dilute 1 L of 20 SSC with 1 L of distilled or deionized water to make 2 L of 10 SSC. 5. Wash Buffer: Add 200 mL of 20 SSC to 1780 mL of distilled or deionized water, plus 20 mL of 10% SDS, to make 2 L of wash buffer. 6. 2 SSC: Dilute 100 mL of 20 SSC with 900 mL of distilled or deionized water to make 1 L of 2 SSC. 8

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 should remain. 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.

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) 10

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 solution just 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-AP from 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 boat gloves 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. 11

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 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).

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 1 3

4 5
6 7 3 2

8
9 10 4

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B.Treat the gels and set up the transfer stacks

Note: The treatment involves four 15-min incubations. During these incubations, prepare the materials for the 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.
28.5 cm

9 cm

9 cm 10 cm 7.5 cm

23 cm

18 cm

18 cm 10 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.
400 ml

Weight (500 g)

Paper towels Nitrocellulose filter or nylon membrane Whatman 3MM paper wicks Transfer buffer Support (inverted casting tray)

Glass plate

Whatman 3MM paper Gel Plastic wrap

Figure 2. Transfer stack

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.

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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 of the 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 gel
The 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 tightfitting 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 Peel off gel leaving what remains of the Figure 3. Use a pencil to pierce the wells stack together as a unit, turn and mark their locations on the membrane. 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). 15

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 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.

F. Prehybridization
1. With gloved hands or with blunt forceps, place the membrane all the way 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 Prehybridization Buffer, 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 before you 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 the membrane 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. 17

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 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 18

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 16,012 7058 17,941 8322 25,183 9024 30,005 13,348 36,374 13,572 40,049 13,689

The probe sequence stretches from base 39,778 to 39,799.

Further Reading
Bloom, 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 21-1215

Name Date

Southern Hybridization Experiment Kit

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 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 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.

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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 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-4chloro-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 Procedures
Note: 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). Figure 1.
1 2 1 3

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.

4 5
6 7 3 2

8
9 10 4

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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 for the 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.
28.5 cm

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.
23 cm

9 cm

9 cm 10 cm 7.5 cm

18 cm

18 cm 10 cm

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.
Paper towels Nitrocellulose filter or nylon membrane Whatman 3MM paper wicks Transfer buffer Support (inverted casting tray)
400 ml

7.5 cm

Weight (500 g)

Glass plate

Whatman 3MM paper Gel Plastic wrap

Figure 2. Transfer stack

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.

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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 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 gel
The 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 the locations 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.

Peel off gel

Figure 3. Use a pencil to pierce the wells and mark their locations on the membrane.

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 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.

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F. Prehybridization
1. With gloved hands or with blunt forceps, place the membrane all the way 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.

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 before you 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 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 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 30,005 7058 36,374 8322 40,049 9024 13,348 13,572 13,689 16,012 17,941 25,183

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