Southern Hybridization

GeNeiTM

Southern Hybridization

GeNeiTM

GeNeiTM Southern Hybridization Teaching Kit Manual

Cat No. KT96 KT96A

Bangalore Genei, 2007 Bangalore Genei, 2007

New Cat No. 106361 106362

Revision No.: 00260905

Southern Hybridization

GeNeiTM

Southern Hybridization

GeNeiTM

CONTENTS
Page No.

Objective Principle Kit Description Materials Provided Procedure Observation & Interpretation Appendix
AGAROSE GEL ELECTROPHORESIS

3 3 4 5 8 12 13

Introduction Principle Procedure

ORDERING INFORMATION

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Objectives:
To learn the technique of Southern Hybridization involving the following experiments: Electrophoretic separation of DNA molecules by agarose gel electrophoresis Electrophoretic transfer of DNA from agarose gel to nylon membrane Immobilization of DNA on to nylon membrane Hybridization and non-isotopic detection of DNA of interest

Principle:
Southern Hybridization technique involves transfer of DNA fragments separated in electrophoretic gels to membrane filters for detection of specific base sequences by complementary probes. Prof. E. M. Southern developed this technique in 1975, hence referred to as Southern transfer. Southern blots are used to identify and quantitate specific DNA sequences, in analysis of genome organization and expression, in the study of genetic diseases, in DNA fingerprinting and analysis of PCR products. In this technique, DNA molecules are size fractionated on a gel and transferred to a nitrocellulose or nylon membrane by capillary or electrophoretic transfer. The DNA is immobilized onto the membrane by UV crosslinking or by baking at 80C. The membrane is washed, prehybridized and then hybridized with a biotin labeled probe. After hybridization, the unbound probe is removed by washes. The membrane is then incubated with the protein block to reduce non-specific interaction. The bound (hybridized) probe is detected by incubating the membrane with streptavidin enzyme conjugate and finally incubated with the substrate solution until sufficient colour (blue) develops. 3 Bangalore Genei, 2007

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Kit Description:
The kit demonstrates the technique of Southern Hybridization. The students will carry out agarose gel electrophoresis by running the DNA digest supplied, transfer the DNA digest electrophoretically onto positively charged nylon membrane, hybridize with biotin labeled probe specific to one of the DNA bands in the DNA digest provided and detect the hybridized DNA by adding streptavidin-HRP (Horse radish peroxidase) conjugate. The final detection is done following addition of substrate, TMB/ H2O2 (Tetramethyl benzidine H2O2 substrate) that reacts with HRP to give a blue coloured DNA band on the nylon membrane. HRP H2O2 TMB + [O] KT96 : H2O + [O] TMBO (Blue)

Materials Provided:
The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival.
Quantity KT96/96A (5 expts.) 100 l 5 x 30 l 50 ml 50 ml 75 ml each 50 ml 15 l 50 ml 2.5 ml 150 ml 15 g 50 l 2.5 g 20 ml 10 Nos. 5 Nos. 1 No.

Materials DNA Marker (Ready to use) Biotinylated Probe Prehybridization Buffer Hybridization Buffer 2X Wash Buffers (A, B, C and D) Blocking Buffer Streptavidin HRP Conjugate Conjugate Dilution Buffer 10X Substrate 10X Electrotransfer Buffer Blocking Powder Tween-20 Agarose 50X TAE Filter Paper Nylon Membrane Petridish
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Store -20C -20C 4C 4C 4C 4C 4C 4C 4C 4C 4C RT RT RT RT RT RT

KT96A :

The kit is designed to carry out 5 Southern Hybridization experiments. The kit also includes electrophoresis equipment and electrotransfer system (ETS-5) required for agarose gel electrophoresis and electrotransfer. The kit is designed to carry out 5 Southern Hybridization experiments.

Note: Electrophoresis equipment and electrotransfer system are required for KT96A. Duration of experiment: Experiment is carried out over a span of two days, approximate time taken on each day is indicated below. Day 1 : 8 hours (Agarose gel electrophoresis, electrotransfer and hybridization) Day 2 : 5 hours (Post Hybridization, Detection and Results) 4 Bangalore Genei, 2007

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Materials Required:
Equipment : Hot air oven, Incubator shaker (45C), UV-transilluminator Reagents : Distilled water, Ethidium Bromide Other Requirements: Crushed ice, Forceps, Gloves, Measuring cylinders, Micropipette, Blade, Scissors, Thermometer, Tips, Transparent polythene sheet.

Note:
Read the entire procedure before starting the experiment. Carry out the experiment by wearing gloves. Specially while handling ethidium bromide agarose gel. Agarose gel to be prepared by addition of ethidium bromide at a concentration of 0.5 g/ml from a stock of 10 mg/ml (refer Agarose Gel Electrophoresis). Dilute the appropriate quantities of buffers (Wash buffers A, B, C and D) to 1X concentration with distilled water just before use. Dilute 10X Electrotransfer buffer to 1X with distilled water. Use autoclaved tips and measuring cylinders to avoid contamination. DNA Marker supplied is ready to use and can be loaded directly onto agarose gel. Re-use the plastic petridish provided for the other four experiments after thorough cleaning with distilled water and drying. Bring the buffers provided to room temperature a day prior to use except for streptavidin HRP- conjugate buffer and substrate solution. Buffers can be prepared according to the required concentration on the previous day of use except for streptavidin HRP- conjugate buffer and substrate solution (which have to be prepared just before use) For preparation of buffers, refer appendix.

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Procedure:
Day 1: Agarose Gel Electrophoresis: 1. Prepare a 1.0% agarose gel containing ethidium bromide. (Refer Agarose Gel Electrophoresis) 2. Load 20 l of ready to use DNA marker supplied onto the gel. Run at 50-100 V until the dye reaches 4.5 cm from the well. 3. Cut the DNA marker lane from the agarose gel as follows: Cut the gel ~ 3 mm above the first band and ~ 2 mm below the last band, ensuring the gel measures about 4 to 4.5 cm. Note: : Mark the appropriate position of the gel to be cut on the gel tank. Take care not to expose yourself to UV light.

Electroblotting: Assemble the electrotransfer apparatus as shown below. Start the arrangement by placing filter paper on the cathode cassette cover followed by the cut gel and nylon membrane. Mark and place the soft side of the nylon membrane to the cut gel. Place the wet filter paper on the nylon membrane followed by anode cassette cover. Tighten the electrotransfer cassette tightly with the screws provided. Note: Ensure no air bubbles are present between any of the layers of filter paper, cut gel and the nylon membrane.
Anode cassette cover Sponge Filter paper Agarose gel Filter paper Sponge Cathode cassette cover

Well Cut

DNA Marker

Nylon Membrane

4. Cut the filter paper (2 nos.) and nylon membrane exactly to the size of the cut gel. Ensure there is no protrusion of the filter paper and the membrane from the gel. 5. Wet the cut gel, nylon membrane, filter papers and the electrotransfer cassette in 1X electrotransfer buffer.

6. Insert the cassette into the apparatus filled with 250 ml of 1X electrotransfer buffer. 7. Connect the cords to power supply according to the convention red: anode, black: cathode and set voltage to 50 V for 3 hours. 8. Turn off the power supply and remove the cassette from the apparatus. Drain the buffer.

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Immobilization of DNA on membrane: 9. Remove the nylon membrane gently from the cassette and place it on a thin transparent polythene sheet and place this on a UV transilluminator (expose the soft side of membrane containing transferred DNA to UV light) with UV lamps switched on for 20 minutes. This helps in fixing the DNA on the membrane. Note: Do not expose yourself to UV light. 10. Turn off the UV transilluminator. Place the membrane in plastic petridish provided and incubate in a hot air oven at 70C for 30 minutes. This ensures complete immobilisation of DNA onto the membrane. Hybridization: 11. Bring the petridish containing the membrane to room temperature after incubation. Add 10 ml of prehybridization buffer to the petridish and incubate at 45C incubator shaker with mild shaking (70-90 rpm) for 45 minutes. 12. After incubation, discard prehybridization buffer taking care not to discard the membrane. 13. Add 10 ml of hybridization buffer to the petridish containing the membrane. 14. Keep 1 vial of biotinylated probe for 10 minutes in a boiling water bath and immediately chill by placing it on ice for 5-10 minutes. Add this probe to the hybridization buffer in the petridish. (Rinse the probe vial with 300 l of hybridization buffer and add it to the petridish.) Incubate the petridish at 45C incubator shaker with mild shaking 70 rpm for 16 hours.

Day 2: Blocking and Detection: 15. Decant the hybridization buffer, add 10 ml of 1X wash buffer A and gently swirl the petridish for 5 minutes at room temperature. Repeat the washes twice (each wash of 5 minutes). Discard the buffer after each wash. 16. Add 10 ml of 1X prewarmed (70C) wash buffer B. and gently swirl the petridish. Incubate at 70C for 5 minutes in a hot air oven & gently swirl. Repeat the washes for another two times. Discard the buffer after each wash. 17. Add 10 ml of 1X blocking buffer to the petridish and incubate at room temperature for 1 hour with gentle rocking. 18. Discard the blocking buffer. 19. Add 9 ml of diluted HRP-streptavidin conjugate to the petridish and incubate at room temperature for 20 minutes with gentle rocking. Discard the conjugate buffer. 20. Add 10 ml of 1X wash buffer C to the petridish and incubate at room temperature for 5 minutes with gentle rocking. Repeat the washes two more times. Discard the buffer after each wash. 21. Add 10 ml of 1X wash buffer D to the petridish and incubate at room temperature for 5 minutes with gentle rocking. Repeat the washes two more times. Discard the buffer after each wash. 22. Add 5 ml of 1X substrate solution and gently swirl at room temperature for 15-20 minutes until a blue colour band develops. 23. After blue colour band is seen, stop the reaction by placing the membrane in distilled water.

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Observation:
Observe for a single blue band on the nylon membrane.

Appendix:
For every experiment, following volumes of reagents and buffers are to be prepared just before use. a. b. c. d. Preparation of Prehybridization buffer: Add 1 g of blocking powder to 10 ml of prehybridization buffer Preparation of Hybridization buffer: Add 1 g of blocking powder to 10 ml of hybridization buffer Preparation of Blocking buffer: Add 1 g of blocking powder to 10 ml of blocking buffer Preparation of Wash buffers A, B, C and D: Thaw and mix the buffer provided thoroughly. Dilute 15 ml each of 2X buffers supplied with 15 ml of distilled water to give buffers at 1X concentration, 30 ml each. Preparation of Streptavidin HRP-conjugate buffer: Prepare 9 ml of conjugate dilution buffer by adding 9 l of Tween-20 to the conjugate dilution buffer. Dilute 3 l of Streptavidin-HRP conjugate with 9 ml of conjugate dilution buffer for each experiment just before use. Preparation of Substrate solution: Dilute 0.5 ml of substrate solution with 4.5 ml of distilled water to give a final volume of 5 ml substrate solution of 1X concentration. Preparation of Electrotransfer buffer: Dilute 25 ml of 10X electrotransfer buffer with 225 ml of distilled water to give a final volume of 250 ml electrotransfer buffer at 1X concentration.

Fig. 1

Fig. 2

e.

Fig. 1 : DNA marker run on 1.0% agarose gel before transfer Fig. 2 : A single blue band on the nylon membrane after electroblotting, hybridization & detection.

Interpretation:
Hybridization is a technique to identify the DNA of interest from the pool of DNA fragments. In this nonisotopic detection technique a biotinylated probe (sequence complementary to the target DNA) is used. The probe binds to the complementary sequences of the DNA marker. This hybridized complex (probe-target complex) is detected by Strptavidin-HRP conjugate, an enzyme that binds to biotin molecule of the probe. On addition of the substrate solution, the enzyme reacts with the substrate to form the blue coloured band as shown in figure 2. The probe-target complex is seen as the major blue band however 1 or 2 very less intense blue band may be seen due to random nonspecific annealing of the probe to the DNA bound on the membrane. 12 Bangalore Genei, 2007

f.

g.

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Agarose Gel Electrophoresis

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Introduction:
Agarose gel electrophoresis is a procedure used to separate DNA fragments based on their molecular weight and is an intrinsic part of almost all routine experiments carried out in molecular biology. The technique consists of 3 basic steps: Preparation of agarose gel Electrophoresis of the DNA fragments Visualization of DNA fragments

Electrophoresis of DNA fragments: Electrophoresis is a technique used to separate charged molecules. DNA is negatively charged at neutral pH and when electric field is applied across the gel, DNA migrates towards the anode. Migration of DNA through the gel is dependent upon: 1. Molecular size of DNA 2. Agarose concentration 3. Conformation of DNA 4. Applied current Matrix of agarose gel acts as a molecular sieve through which DNA fragments move on application of electric current. Higher concentration of agarose gives firmer gels, i.e., spaces between cross-linked molecules is less and hence smaller DNA fragments easily crawl through these spaces. As the length of the DNA increases, it becomes harder for the DNA to pass through the spaces, while lower concentration of agarose helps in movements of larger DNA fragments as the spaces between the cross-linked molecules is more. The progress of gel electrophoresis is monitored by observing the migration of a visible dye (tracking dye) through the gel. Two commonly used dyes are Xylene cyanol and Bromophenol blue that migrate at the same speed as double stranded DNA of size 5000 bp and 300 bp respectively. These tracking dyes are negatively charged, low molecular weight compounds that are loaded along with each sample at the start of run, when the tracking dye reaches towards the anode, run is terminated.

Principle:
Preparation of Agarose Gel: Agarose is a linear polymer extracted from seaweeds. Its basic structure is shown in the figure .

HO

CH2O
OH

HO
Figure: Basic unit structure of agarose. Purified agarose is a powder insoluble in water or buffer at room temperature but dissolves on boiling. Molten solution is then poured into a mould and allowed to solidify. As it cools, agarose undergoes polymerization i.e., sugar polymers cross-link with each other and cause the solution to gel, the density or pore size of which is determined by concentration of agarose.

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Visualization of DNA fragments: Since DNA is not naturally coloured, it will not be visible on the gel. Hence the gel, after electrophoresis, is stained with a dye specific to the DNA. Discrete bands are observed when there is enough DNA material present to bind the dye to make it visible, otherwise the band is not detected. The gel is observed against a light background wherein DNA appears as dark coloured bands. Alternatively, an intercalating dye like Ethidium bromide is added to agarose gel and location of bands determined by examining the gel under UV light, wherein DNA fluoresces. Note: Ethidium bromide must be handled carefully as it is a mutagen and a carcinogen. Wear gloves while handling EtBr solution & gels stained with EtBr.

Procedure:
Preparation of 1% Agarose Gel 1. Prepare 1X TAE by diluting appropriate amount of 50X TAE buffer. (For one experiment, approximately 200 ml of 1X TAE is required. Make up 4 ml of 50X TAE to 200 ml with distilled water). 2. Weigh 0.5 g of agarose and add to 50 ml of 1X TAE. This gives 1% agarose gel. 3. Boil till agarose dissolves completely and a clear solution results. 4. Meanwhile place the combs of electrophoresis set such that it is approximately 2 cm away from the cathode. 5. Pour the agarose solution in the central part of tank when the temperature reaches approximately 60C. Do not generate air bubbles. The thickness of the gel should be around 0.5 to 0.9 cm. Keep the gel undisturbed at room temperature for the agarose to solidify. 6. Pour 1X TAE buffer into the gel tank till the buffer level stands at 0.5 to 0.8 cm above the gel surface. 7. Gently lift the combs, ensuring that wells remain intact.

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Electrophoresis 8. Connect the power cord to the electrophoretic power supply according to the convention red: anode, black: cathode. 9. Load the samples in the wells in the desired order. 10. Set the voltage to 50 V and switch on the power supply. 11. Switch off the power when the tracking dye (bromophenol blue) from the well reaches th of the gel. This takes approximately one hour. Staining Procedure to Visualize DNA 12. Prepare 1X staining dye by diluting 6X dye (1:6) with distilled water. (Approximately 50 ml of 1X staining dye is required for one experiment. Therefore, make up 8 ml of 6X dye to 48 ml with distilled water). 13. Carefully transfer the gel (from gel tank) into a tray containing 1X staining solution. Make sure that the gel is completely immersed. 14. For uniform staining, place the tray on a rocker for approximately one hour or shake intermittently every 10 to 15 minutes. 15. Pour out the staining dye into a container. (The dye can be reused twice). Destain the gel by washing with tap water several times till the DNA is visible as a dark band against a light blue background. Note: Alternatively, Ethidium bromide can be used for visualizing DNA fragments. Add Ethidium bromide to molten agarose to a final concentration of 0.5 g/ml (from a stock of 10 mg/ml in water), when temperature is around 50C. Mix and cast the gel. After electrophoresis, DNA samples can be visualized under UV light, they appear fluorescent. No destaining is required in this case.
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Ordering Information
Product GeNeiTM Southern Hybridization Teaching Kit (Consumables for 5 experiments & Elpho Kit (ETS 5)) GeNeiTM Southern Hybridization Teaching Kit Consumables for 5 experiments) Size 1 Pack Cat # KT96

1 Pack

KT96A

Email: Sales: geneisales@sanmargroup.com Customer Support: geneitechsupport@sanmargroup.com

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Note:

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