DNA HYBRIDIZATION AND DETECTION BY CHEMILUMINESCENCE

PROCEDURE FOR SOUTHERN TRANSFER AND CHEMILUMINESCENT DETECTION

Principle

The principle of the Southern transfer is to transfer the DNA from the agarose gel onto a membrane. The DNA on the membrane is then hybridized with a probe that is labeled with biotin. The probe specifically binds to complementary DNA on the nylon membrane. The degree of hybridization of a complimentary probe is a measure of the amount of the specific target sequence in the sample. This is detected via a streptavidin alkaline phosphatase conjugate. Upon enzymatic dephosphorylation, the substrate decomposes, resulting in a prolonged, constant emission of light that is the imaged on X-ray film or image analyzer.

Sample

Agarose gel.

Reagents (see compositions at the end of the page)

0.5M NaOH, 1.5M NaCl

1M Tris-HCl (pH 8.0), 1.5M NaCl.

20X SSC

Biotin labeled probe

DNA Hybridization buffer

20% SDS

Sterile water

10 X PBS

CPD-Star Ready to Use Substrate for Alkaline Phosphatase (Tropix catalog #MS250R)

I-Block Protein Based Blocking Reagent (Tropix catalog #AB100)

Avidx-AP Streptavidin-AP Conjugate (Tropix catalog #APA10)

Sheared salmon Sperm DNA (10 mg/ml)

0.25N HCl

Blocking buffer

Wash buffer

Assay buffer

Materials and Equipment

Pipettors and tips

Glassware

0.5 ml microcentrifuge tubes, autoclaved

Shaking water bath, 60oC

Hybridization bags

Heat sealer

Plastic trays

Nylon membrane

Filter paper

Stratagene Stratalinker 1800

X-Ray film and cassette

0.45uM syringe filter

65oC water bath

70% ethanol

Procedure

Southern Transfer:

NOTE: Use at least twice volume of the gel of all solutions (0.5 M HCl, 0.5 M NaOH, 1.5 M NaCl and 1 M Tris-HCl pH 8.0, 1.5 M NaCl). For example, if you used 300 ml of agarose solution to pour the gel, use 600 ml of each of these solutions
      1. After visualization in Ethidium bromide, denature the gel for 45 minutes in 0.5 M NaOH, 1.5 M NaCl. NOTE: For high molecular weight DNA, put the gel first into 0.25 N HCl for 15 minutes, pour off and add the 0.5 M NaOH, 1.5 M NaCl for 10 minutes then pour off and add fresh 0.5 M NaOH, 1.5M NaCl for 45 minutes.
      2. Neutralize the gel for 15-20 minutes in 1M Tris-HCl (pH 8.0), 1.5 M NaCl.
      3. Transfer by capillary method:
      4. Pour approximately 750 ul of 6X SSC into a tray. Put a glass plate large enough for the gel to fit on top of the tray. Cut a piece of filter paper wider than the gel and long enough to touch the bottom of the tray on both sides. Wet the piece of filter paper in 6X SSC and place it on top of the glass plate, making sure that there are no bubbles under the paper.
      5. Place the gel upside down on the filter paper noting orientation. Place plastic wrap approximately 2 mm over the top of all edges of the gel so that filter paper placed on top of the gel will not come into contact with the filter paper under the gel.
      6. Wet a piece of nylon paper in 6X SSC and place it on top of the gel. Make sure there are no bubbles.
      7. Wet in 6X SSC and place 2 pieces of filter paper also the same size as the gel on top of the nylon membrane and make sure there are no bubbles.
      8. Place 2 stacks of paper towels (approximately 8 cm each) on top of the filter paper so that the gel and filter paper are covered evenly by the paper towels. (The 2 stacks should touch in the center of the gel and extend out to cover it entirely).
      9. Make sure that the stacks of paper towels are equal and even and place a glass plate centered on top of the paper towels. Place a 0.5 L bottle full of water in the center of the glass plate to distribute the weight evenly across the gel and membrane.
      10. Let transfer by capillary method over night.
      11. When the transfer is complete, rinse the membrane in 2X SSC, then UV irradiate in the Stratagene Stratalinker 1800, using the automatic mode.
      12. Dry the nylon at 37oC over night, wrap in plastic wrap and store in refrigerator, or use immediately for hybridization.

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Hybridization
      1. Denature the salmon sperm DNA. Add 100 ul of denatured salmon sperm DNA per 10 mL of hybridization buffer. *This is what you use for the hybridization.
      2. Denature the biotin labeled probe. Use 10 ng of probe per ml of buffer to be used. (Probes are 1 ug/100 ul). Cool the probes rapidly on ice. Hybridization is done in a hybridization bag. 100 ul of DNA Hybridization buffer is needed per cm2 of nylon.
      3. Pipet the Hybridization buffer into the hybridization bag. Dilute the probe in 100 ul - 200 ul of hybridization buffer. Cut a corner off of the bag and pipet the diluted, denatured probe into the bag. Heat-seal the corner. Mix the contents well. Cut a corner off of the bag and expel all of the bubbles.
      4. Seal the bag and incubate in a 65oC water bath for 3 - 24 hours.
      5. While the DNA is hybridizing, prepare the following buffers:

        1. 0.2X SSC, 1% SDS

        6. 2. 0.1X SSC, 1% SDS

        3. 0.1X SSC

        4. Blocking buffer

        5. Wash buffer

      6. Assay buffer

Washing
    1. When hybridization is complete, remove the hybridization bag from the water bath.
    2. Wash the nylon 2 X 5 minutes at room temperature in 0.2X SSC, 1% SDS.
    3. Wash 2 X 15 minutes at 60oC (in shaking water bath), in 0.1X SSC, 1% SDS.
    4. Wash 2 X 5 minutes at room temperature in 0.1X SSC.

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Membrane blocking and chemiluminescent detection:
      1. Wash the blot 2 X 5 minutes at room temperature in Blocking buffer.
      2. Incubate for 10 minutes at room temperature in Blocking buffer.
      3. Dilute the Avidx-AP conjugate 1:5,000 in Blocking buffer. Use 2.0 ul of Avidx-AP conjugate in 10 mL of Blocking buffer per 100 cm2 of nylon in hybridization bag.
      4. Incubate the blot on rocker for 20 minutes at room temperature.
      5. Wash 1 X 5 minutes in Blocking buffer.
      6. Wash 3 X 5 minutes in Wash buffer.
      7. Wash 2 X 2 minutes in Assay buffer.
      8. Put the nylon into a new hybridization bag and add the Chemiluminescent Substrate Solution to the bag (5 ml/100 cm2). *In the dark* and rock for 5 minutes.
      9. Squeeze the excess substrate solution off of the membrane by rolling a pipet over the plastic.
      10. Take the blot out of the bag and put it between 2 clean pieces of plastic. Imaging is done using X-Ray film or by Kodak Image analyzer.

References

Protocol for Southern-Light Chemiluminescent Detection system for Biotin Labeled Probes, Tropix, Inc.,

Sambrook, Fritsch, Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, 1989, Pgs. 9.31 - 9.46.

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BUFFERS

0.5 M NaOH, 1.5 M NaCl

COMPOSITION TO MAKE 1L USE:

20 g NaOH

87.6 g NaCl

Dissolve NaOH and NaCl in dH20. Bring up to a final volume of 1L. Store at room temperature.

1 M TRIS pH 8.0, 1.5 M NaCl

COMPOSITION TO MAKE 1L USE:

Tris 121.1 g

NaCl 87.6 g

HCl

Dissolve Tris and NaCl in approximately 700 ml of dH20. pH to 8.0 with HCl (it takes approximately 45 ml). Bring up to a final volume of 1L. Store at room temperature.

BLOCKING BUFFER

COMPOSITION TO MAKE 300ML USE:

0.2% I-Block Reagent or dry milk

10 X PBS

0.1% SDS

For blocking buffer: Add 30 ml 10X PBS to 240 ml of sterile water and 0.6 g dry milk or I-block reagent. Heat the mixture in a microwave oven until it starts to boil. (It is very important to boil it for at least 10 seconds, otherwise a background will develop in the Southern blot). Add 30 ml of a 1% SDS solution and mix. Cool it down to RT before use.

WASH BUFFER

COMPOSITION TO MAKE 500 ml USE:

50 ml 10X PBS

12.5 ml of 20% SDS

Add sterile water to 500 ml.

ASSAY BUFFER

COMPOSITION TO MAKE 1L USE:

9.6 ml Diethanolamine

0.2 g MgCl2

Dissolve DEA in 800 ml of sterile water and adjust pH to 10.0 with HCl. Add MgCl2. Bring volume up to 1 L with sterile water. Store in refrigerator.

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DNA HYBRIDIZATION BUFFER

COMPOSITION TO MAKE 100ML USE:

10 g PEG

10 ml 5 M NaCl

4 ml 0.5 M Na phosphate (pH 7.0)

1 ml 0.5 M EDTA

5 ml 10% SDS

Combine everything except for the SDS in 50 ml of dH20. Heat in the microwave to dissolve the PEG if necessary. Adjust the volume to 95 ml with dH20. Autoclave. Add the SDS and mix. Store at room temperature for up to 2 months.

10X PHOSPHATE BUFFERED SALINE (PBS)

COMPOSITION TO MAKE 1L USE:

2 g KCl

2 g KH2PO4

80 g NaCl

11.45 g Na2HPO4

Dissolve everything in approximately 800 ml of dH20. pH should be 7.2 without adjusting. Autoclave and store at room temperature.

NOTE: If Na2HPO4 (7H20), use 21.6 g, or if Na2HPO4 (12H20), use 28.8 g.

20 X SSC

COMPOSITION TO MAKE 1L USE:

175.3 g NaCl

88.2 g Na Citrate dihydrate

HCl

Dissolve in approximately 800 ml dH20. Adjust to pH 7.0 with dilute HCl. Bring up to a final volume of 1L and autoclave. Store at room temperature.