Unzipping Genes

Product Information

HiPer® Southern Blotting Teaching Kit

Product Code: HTBM027

Number of experiments that can be performed: 5
Duration of Experiment: 2 days
Day1: Agarose Gel Electrophoresis, Electro transfer and
Hybridization
Day2: Detection and Result

Storage Instructions:
 The kit is stable for 12 months from the date of manufacture
 Store DNA Sample and Biotinylated Probe at -20 oC
 Store Tween 20, Agarose, 10XTBE, Nylon Membrane and Petri plate at
room temperature (15-25 oC)
 Store the rest of materials at 2-8oC

Registered Office : Commercial Office

WHO 23, Vadhani Industrial Estate,LBS Marg, A-516, Swastik Disha Business Park, Tel: 00-91-22-6147 1919
15 Mumbai - 400 086, India. Fax: 6147 1920, 2500 5764
GMP Tel. : (022) 4017 9797 / 2500 1607
Via Vadhani Indl. Est., LBS Marg,
Mumbai - 400 086, India Email : [email protected]
CERTIFIED Fax : (022) 2500 2286 Web : www.himedialabs.com

The information contained herein is believed to be accurate and complete. However no warranty or guarantee whatsoever is made
or is to be implied with respect to such information or with respect to any product, method or apparatus referred to herein
 Index

Sr. No. Contents Page No.

1 Aim 3

2 Introduction 3

3 Principle 3

4 Kit Contents 5

5 Materials Required But Not Provided 5

6 Storage 5

7 Safety 5

8 Important Instructions 6

9 Procedure 6

10 Flowchart 8

11 Observation and Result 9

12 Interpretation 9

13 Troubleshooting Guide 9

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Aim:

To learn the technique of Southern Blotting for the detection of a specific DNA fragment

Introduction:

Southern blotting or Southern hybridization is a widely used technique in molecular biology for transfer of DNA
molecules; usually restriction fragments, from an electrophoresis gel to a nitrocellulose or nylon membrane, and is
carried out prior to detection of specific molecules by hybridization probing. In this method a DNA mixture is
separated by agarose gel electrophoresis according to their size followed by transfer of the DNA bands to
nitrocellulose/nylon membrane. Finally, the DNA of interest is probed for a specific sequence.

Principle:

Southern hybridization, also called Southern blotting, is a commonly used method for the identification of DNA
fragments that are complementary to a known DNA sequence. It allows a comparison between the genome of a
particular organism and that of an available gene or gene fragment. This technique also tells us whether an organism
contains a particular gene, and provides information about the organism and restriction map of that gene. Southern
hybridization was named after its inventor, Edward M. Southern, who developed the technique in 1975. As a result
subsequent blotting techniques have used similar nomenclature, for example Northern blotting, the transfer of RNA;
Western blotting, the transfer of proteins; and Southwestern blotting, for the characterization of proteins that bind
DNA. In Southern Blotting the chromosomal DNA is isolated from an organism of interest, and digested with
restriction enzyme. The restriction digested fragments are electrophoresed on an agarose gel, which separates the
fragments on the basis of size. The next step is to transfer fragments from the gel onto nitrocellulose filter or nylon
membrane. This can be performed either by electrotransfer i.e. electrophoresing the DNA out of the gel and onto a
membrane or by the simple capillary method. The transfer or a subsequent treatment results in immobilization of
the DNA fragments, so the membrane carries a semi permanent reproduction of the banding pattern of the gel. The
DNA is bound irreversibly to the membrane by baking at high temperature (80°C) or by UV crosslinking. For the
detection of a specific DNA sequence, a hybridization probe is used. A hybridization probe is a short (100-500bp),
single stranded nucleic acid that will bind to a complementary piece of DNA. Hybridization probes are labeled with
a marker (radioactive or non-radioactive) so that they can be detected after hybridization. In non-radioactive
detection the probe is labeled with biotin or dioxigenin. The membrane is washed to remove non-specifically bound
probe and the hybridized probe is detected by treating the membrane with a conjugated enzyme, followed by
incubation with the chromogenic substrate solution. As a result a visible band can be seen on the membrane where
the probe is bound to the DNA sample. The entire procedure can be divided into following steps:

I. Agarose Gel Electrophoresis: Agarose gel electrophoresis is a technique for separation of DNA molecules
according to their molecular size. This is achieved when negatively charged nucleic acids migrate through an
agarose gel matrix under the influence of an electric field (electrophoresis). Shorter molecules move faster and
migrate farther than the larger ones. The position of DNA in the agarose gel is visualized by staining with low
concentration of fluorescent intercalating dyes, such as Ethidium bromide.

II. Southern Blotting: Southern blotting is the electro transfer/capillary transfer of resolved DNA fragments from
the agarose gel to the nitrocellulose/nylon membrane. For this transfer procedure, the gel is placed on the membrane
and both of them are sandwiched between two filter papers as shown in Figure 1:

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 Filter Paper
Agarose Gel

Nylon Membrane

Filter Paper

Fig 1: Arrangement of the gel and membrane for transfer

During electro transfer the DNA bands are transferred to positively charged nylon membrane in the presence of a
specific buffer. First transfer the set (as shown in Fig 1) between two sponge pads and then place it in a plastic
cassette. The entire set is then placed inside a gel tank filled with transfer buffer. The resolved DNA fragments are
transferred to the corresponding positions on the nylon membrane after the electro transfer. The DNA of interest is
detected on the membrane.

III. Detection:

After electrotransfer, DNA bands bound to the membrane are detected chromogenically. A suitable blocking reagent
is used to block the unoccupied sites on the membrane. Then the DNA of interest is hybridized with a biotinylated
probe specific to it. The membrane is washed to remove excess unbound probe. It is then treated with Horseradish
peroxidase (HRP)-conjugated streptavidin which attaches to the hybridized DNA. Finally, the membrane is incubated
in a substrate solution containing TMB/ H2O2 (Tetramethyl benzidine H2O2 substrate) that reacts with HRP and as a
result a blue coloured DNA band develops on the nylon membrane as shown in Figure 2.

Developed blue
TMB colour
Streptavidin-HRP
Biotin

Hybridization with Development of blue colour after

Biotinylated probe reaction with Streptavidin-HRP and
TMB

Fig 2: The hybridized DNA is detected after treatment with Streptavidin-HRP, followed by TMB substrate

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Kit Contents:

This kit can be used for the detection of a specific DNA band in a given sample.
Table 1: Enlists the materials provided in this kit with their quantity and recommended storage

Product Quantity
Sr. No. Materials Provided Storage
Code 5 expts
1 TKC314 DNA Sample 0.06 ml -20oC
2 TKC315 Prehybridisation Buffer 60 ml 2-8oC
3 TKC316 Hybridisation Buffer 60 ml 2-8oC
4 TKC317 Biotinylated Probe 5 x 0.015 ml -20oC
5 TKC318 2X Wash Buffer I 80 ml 2-8oC
6 TKC319 2X Wash Buffer II 80 ml 2-8oC
7 TKC320 2X Wash Buffer III 80 ml 2-8oC
8 TKC321 2X Wash Buffer IV 80 ml 2-8oC
9 TKC322 Blocking Buffer 60 ml 2-8oC
10 TKC323 Blocking Powder 2g 2-8oC
11 TKC324 10X Transfer Buffer 200 ml 2-8oC
12 TKC143 TMB/H2O2 30 ml 2-8oC
13 TKC325 Streptavidin HRP Conjugate 0.040 ml 2-8oC
14 TKC326 Conjugate Dilution Buffer 50 ml 2-8oC
15 MB067 Tween 20 0.05 ml RT
16 MB002 Agarose 3g RT
17 ML011 10X TBE 300 ml RT
18 TKC327 Nylon Membrane with filter paper 5 Nos. RT
19 PW001 Sterile Disposable Petriplates 2 Nos. RT

Materials Required But Not Provided:

Glassware: Test tubes

Reagents: Distilled water, Ethidium bromide (10 mg/ml), deionized water
Other requirements: Gel transfer apparatus, Gel rocker, Micropipettes, Tips, Microwave/Burner/Hotplate,
Hot Air Oven, Incubator Shaker (45oC), Forceps

Storage:

HiPer® Southern Blotting Teaching Kit is stable for 12 months from the date of manufacture without showing any
reduction in performance. Store all the reagents as mentioned above.

Safety:

Precaution: UV light can damage the eyes and skin. Always wear suitable eye and face protection when
working with a UV light source. UV light damages DNA. If DNA fragments are to be extracted from the gel,
use a lower intensity UV source if possible and minimize exposure of the DNA to the UV light.
Disposal of Ethidium bromide waste: All items that were in contact with Ethidium bromide must be disposed

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off in the designated waste container (marked with "Ethidium bromide waste") within the Gel-Doc-Area, including
gels, tissue paper to clean UV table, and nitrile gloves.
Hazard: Ethidium bromide is a powerful mutagen and is very toxic. Appropriate safety precautions should be taken
by wearing latex gloves; however, use of nitrile gloves is recommended.
Important Instructions:

1. Read entire procedure carefully before starting the experiment.

2. DNA sample provided is ready to use and can be loaded directly onto the agarose gel.
3. Preparation of Prehybridisation Buffer: Add 0.1g of blocking powder to 10 ml of prehybridisation
buffer. Mix well before use.
4. Preparation of Hybridization Buffer: Add 0.1g of blocking powder to 10 ml of hybridization buffer.
Mix well before use.
5. Preparation of 1X Transfer Buffer: To prepare 1 litre of 1X Transfer Buffer, take 100 ml of 10X
transfer buffer and add 900 ml sterile distilled water*. Store at 2-8oC. Mix well before use. The 1X
Transfer Buffer can be reused 2-3 times.
6. Preparation of 1X Wash Buffer I: Dilute 15 ml of 2X Wash Buffer I with 15 ml of autoclaved deionized
water. Mix well.
7. Preparation of 1X Wash Buffer II: Dilute 15 ml of 2X Wash Buffer II with 15 ml of autoclaved
deionized water. Mix well.
8. Preparation of 1X Wash Buffer III: Dilute 15 ml of 2X Wash Buffer III with 15 ml of autoclaved
deionized water. Mix well.
9. Preparation of 1X Wash Buffer IV: Dilute 15 ml of 2X Wash Buffer IV with 15 ml of autoclaved
deionized water. Mix well.
10. Preparation of Blocking Buffer: Add 0.1g of blocking powder to 10 ml of blocking buffer
11. Preparation of Streptavidin HRP-Conjugate Buffer: Add 9 μl of Tween 20 in 9 ml of conjugate
dilution buffer. Add 6.0 μl of Streptavidin- HRP Conjugate in 9 ml of conjugate dilution buffer for
each experiment just prior to use.
12. Bring the buffers provided to room temperature a day prior to use except for Streptavidin-HRP
conjugate buffer and TMB/H2O2
13. Ensure no air bubbles are present between any of the layers of filter paper, cut gel and nylon
membrane during electroblotting.
14. Reuse the plastic petri plate 2-3 times after thorough cleaning with distilled water and drying.

Procedure:

Day 1: Agarose Gel Electrophoresis

1. Preparation of 1X TBE: To prepare 500 ml of 1X TBE buffer, add 50 ml of 10X TBE Buffer to 450 ml of
sterile distilled water*. Mix well before use.

2. Preparation of agarose gel: To prepare 50 ml of 1 % agarose gel, add 0.5 g agarose to 50 ml 1X TBE
buffer in a glass beaker or flask. Heat the mixture on a microwave or hot plateswirling the glass beaker/
flask occasionally, until agarose dissolves completely (Ensure that the lid of the flask is loose to avoid
buildup of pressure). Allow solution to cool to about 55-60oC. Add 0.5 μl Ethidium bromide, mix well and
pour the gel solution into the gel tray. Allow the gel to solidify for about 30 minutes at room temperature.

3. Loading of the DNA samples: To prepare sample for electrophoresis, load 10 μl of DNA sample into the well.

4. Electrophoresis: Connect the power cord to the electrophoretic power supply according to the conventions:
Red-Anode and Black- Cathode. Electrophorese at 100-120 volts and 90 mA until dye markers have migrated an

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 appropriate distance, depending on the size of DNA to be visualized.

5. Cut the DNA Marker from the agarose gel by viewing it under UV transilluminator. Cut the gel about 3 mm
from the first band and 2 mm below the last band ensuring the gel measures about 4 to 4.5 cm.

* Molecular biology grade water is recommended (Product code: ML024).

6. Take out the nylon membrane with the filter paper (provided). Ensure there is no protrusion of the filter
paper and membrane from the gel.

7. Wet the cut gel, nylon membrane, filter papers and the electrotransfer cassette in 1X Electrotransfer buffer.

Electroblotting:

1. Assemble the gel with nylon membrane and filter papers as shown in figure 1. This blotting sandwich is
placed within the sponge and blotting cassette. Try to avoid air bubble between gel and nylon membrane by
rolling a glass tube on the membrane.
Note: Take out the transparent sheets carefully while using the nylon membrane.
2. Insert this cassette into the gel transfer apparatus filled with cold transfer buffer and then connect the
transfer unit to power supply as per conventions.
3. Electrophoreses the sample at 100V, 90 mA for 2 hours for blotting.

Immobilization of DNA on membrane:

1. Remove the nylon membrane after transfer from the blotting cassette and place the membrane in petri plate on a
UV transilluminator (expose the membrane containing transferred DNA to UV light) for 20 minutes. This helps in
fixing the DNA on the membrane.
2. Turn off the UV transilluminator and incubate the plate containing membrane in hot air oven at 70 oC for 30
minutes. This ensures complete immobilization onto the membrane.

Hybridization:

1. Bring the petri plate containing membrane to room temperature after incubation. Add 10 ml of prehybridisation
buffer into it and incubate at 45 oC incubator shaker with mild shaking at 70-90 rpm for 45 minutes.
2. After incubation, discard the prehybridisation buffer. Care should be taken not to discard the membrane.
3. Add 10 ml of hybridisation buffer to the petri plate containing membrane.
4. Keep 1 vial of biotinylated probe for 10 minutes in boiling water bath and immediately chill by placing it on ice
for 5-10 minutes. Add 15 μl of this probe to the hybridization buffer in the petri plate.
5. Incubate the petri plate at 45 oC incubator shaker with mild shaking at about 70-90 rpm for 16 hours.

Day 2: Blocking and Detection:

1. Decant the hybridization buffer, add 10 ml of 1X Wash Buffer I and gently swirl the petri plate for 5 minutes at
room temperature. Repeat the washes twice (each wash for 5 minutes). Discard the buffer after each wash.
2. Add 10 ml of prewarmed 1X Wash Buffer II (70 oC) and gently swirl the petri plate. Incubate at 70 oC for 5 minutes
in a hot air oven and gently swirl. Repeat the washes for another 2 times. Discard the buffer after each wash.
3. Add 10 ml of blocking buffer to the petri plate and incubate at room temperature for 1 hour with gentle rocking.
Discard the blocking buffer.
4. Add 9 ml of diluted Streptavidin-HRP conjugate buffer to the petri plate and incubate at room temperature for 20
minutes with gentle rocking. Discard the conjugate buffer.

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5. Add 10 ml of 1X Wash Buffer III to the petri plate and incubate at room temperature for 5 minutes each with
gentle rocking. Repeat the washes two more times. Discard the buffer after each wash.
6. Add 10 ml of 1X Wash Buffer IV to the petri plate and incubate at room temperature for 5 minutes each with
gentle rocking. Repeat the washes two more times. Discard the buffer after each wash.
7. Add 5 ml of TMB/H2O2 and gently swirl at room temperature for 15-20 minutes until a blue colour band
develops.
8. After blue colour band is seen stop the reaction by placing the membrane in distilled water.

Flow chart:
DNA (Restriction
digested fragments)

DNA bands fractionated on

agarose gel

Electro transfer of DNA from

gel to membrane

Hybridization of membrane with

biotinylated probe

Detection of band after

treatment with TMB

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Observation and Result:
1 2

Fig 3: Gel image and immunoblot of the DNA sample after Agarose Gel Electrophoresis
and Southern blotting

Lane 1: DNA sample after 1 % agarose gel electrophoresis

Lane 2: After Southern hybridization a blue band develops on the nylon membrane

Interpretation:

In non-radioactive Southern Hybridization a biotinylated probe is hybridized to the complementary target DNA. The
biotin of resulting hybridized complex binds to Streptavidin-HRP conjugate. In presence of TMB substrate HRP
reacts with it and forms a blue colour which appears as a blue band on the nylon membrane.

Troubleshooting Guide:

Sr.No Problem Probable Cause Solution

Following DNA transfer to membrane, view the gel
Low Sensitivity Incomplete transfer
with UV transilluminator to see if any DNA has
(faint bands
1 remained in the gel
or no bands)
Target DNA not effectively fixed Check UV lamp or oven temperature
on membrane
Insufficient washing or contamination Wash the membrane thoroughly as mentioned in
in buffer the brochure
Always clean forceps after they are used with
2 High
hybridization solutions containing labeled probe.
background
Contaminated forceps Dirty forceps may deposit dye on membrane that
will not wash away

Probe added onto membrane Always add the probe to hybridization solution

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Technical Assistance:

At HiMedia we pride ourselves on the quality and availability of our technical support. For any kind of technical
assistance mail at [email protected]

25°C Storage temperature

15°C

Do not use if package is

damaged

HiMedia Laboratories Pvt. Limited,

23 Vadhani Industrial Estate,
LBS Marg,Mumbai-86,MS,India

PIHTBM027_0/0419 HTBM027-06

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