Southern (DNA) and Northern (RNA) Hybridizations

with MAGNA and MagnaGraph Nylon Membranes

Gel Preparation

Southern
Run DNA on an agarose gel with a running buffer of TAE or TBE. Rinse gel with DI water. If necessary, fragment DNA by immersing the gel in 0.25 N HCl for 8-10 minutes. Denature DNA by soaking gel in 1.0 M NaCl/0.5 M NaOH two times for 20 minutes each. Neutralize the gel by soaking in 0.5 M Tris/pH 7.5, 1.5 M NaCl two time for 20 minutes each.

Northern
Run RNA under denaturing conditions in a Glyoxal, Formaldehyde, or Methyl Mercuric Hydroxide gel. Gel should be 0.8-1.5% agarose, 2.5-5.0mm thick. Stain with 33 µg/ml acridine orange in 10 mM NaPo₄/pH 6.5, then destain 3 x 15 minutes in buffer. Or, stain with ethidium bromide, 1 µg/ml in 50 mM NaOH for 25 minutes, then destain in 200 mM NaOAc/pH 4.0, 2 x 20 minutes.

Membrane Preparation

Float membrane on distilled water, then immerse until thoroughly wet. Soak the membrane in the transfer buffer until use.

Capillary Transfer

Use a transfer buffer of 10 x SSPE or SSC.

Cut three pieces of filter paper (chromatography grade) three inches longer than the glass plate to be used for the capillary transfer. Saturate the filter paper with transfer buffer and place on top of a glass plate.

Place the gel on top of the filter paper, and the membrane over the gel. Place 5 pieces of filter paper cut to the size of the gel over the assembly. Throughout the transfer, do not allow the paper on top of the gel to contact the paper below the gel. This is done by placing strips of Parafilm around the sides of the gel.

Place the glass plate and the gel assembly on top of a glass baking tray filled with transfer buffer. Allow the bottom layer of filter paper to overhang into the transfer solution in the glass baking tray. Place a 2 inch stack of paper towels on top of the gel assembly and secure it with a light weight. Make sure that the filter paper under the gel is completely saturated. The wicking action of the solution through the gel and up the paper towels allows the solution to transfer the DNA or RNA molecules to the membrane.

Secure plastic wrap over the entire assembly and place in a cold room for 3 hours to overnight. If the paper towels become saturated with transfer buffer, replace them with dry ones. After transfer, stain the gel with 0.5 µg/ml ethidium bromide to check transfer efficiency.

Alternative Transfer Systems

Vacuum blotting, semi-dry electroblotting, bi-directional transfers, and positive pressure blotting systems can all be used with GE Osmonics nylon membranes. Follow manufacturers instructions, and contact GE Osmonics Technical Services with any questions.

Immobilization

After blotting, wash the membranes in 5 x SSPE at 60°C for 5 minutes. This is an optional step to remove residual agarose. The membrane can be immobilized by baking or UV crosslinking.

To bake the membrane a vacuum oven or convection oven may be used, place the membrane in a 65°-80°C oven for 1 hour or until the membrane is completely dry.

The membrane may be UV crosslinked by exposing the membrane to a controlled UV source. Follow the instructions of the manufacturer of the crosslinker or expose a damp membrane to a transilluminator. The total exposure should be 120mJ/cm. Increased exposure will cause a decrease in signal intensity after reprobing, proportional to the amount of overexposure.

Hybridization Procedure

Hybridization is most commonly done in heat-sealable bags in order to conserve solution and protect researchers from exposure to radioactivity. All hybridization solutions should be filtered before use with a Cameo 25AS, 0.22 µm supported cellulose acetate filter (GE Osmonics catalog #DDA02025S0). (NOTE: Use only cellulose acetate filters; other membrane types may not perform comparably). The low binding Cameo 25AS will filter prehybridization and hybridization solutions without nonspecifically binding essential components of these solutions.

Prehybridization

This step should always be carried out at the temperature of the hybridization. Place the membrane in a heat-sealable bag without the probe in 0.25 ml/cm² of the following prehybridization buffer:

Southern Prehybridization Solution		Northern Hybridization Solution
6 x SSPE 5 x Denhardt's solution 0.5-1.0% SDS 50 µg/ml denatured DNA 10% Dextran Sulfate		5 x SSPE 50% Formamide 0.1-0.5% SDS 100 µg/ml denatured DNA 5 x Denhardt's solution Shake one to two hours, at 42°C.

Hybridization

Remove prehybridization solution completely from bag, and add the hybridization solution.

Hybridization temperature should be determined by the presence of formamide in the hybridization solution.

Hybridization Temperature Chart

Temperature		% Formamide		Hybridization Solution
42°C		50%		Low Temperature Hybridization Solution
65°C		0%		High Temperature Hybridization Solution

Low Temperature Hybridization Solution

Southern		Northern
50% formamide (47%) 5 x Denhardt's solution 6 x SSPE 0.2% SDS 100 µg/ml denatured DNA 10% dextran sulfate		50% formamide (47%) 5 x Denhardt's solution 5 x SSPE 0.2% SDS 100 µg/ml denatured DNA 10% dextran sulfate

High Temperature Hybridization Solution

Southern		Northern
5 x Denhardt's solution 6 x SSPE 0.5% SDS 50 µg/ml denatured DNA 10% dextran sulfate		5 x Denhardt's solution 5 x SSPE 0.5% SDS 100-200 µg/ml denatured DNA 10% dextran sulfate

NOTE: When using nylon membranes, the potential for backgrounds is greater. Increased volumes of Denhardt's solution and SDS can help further block the membrane.

Dextran sulfate is a rate enhancer for probes larger than 200 base pairs and should not be used with oligonucleotide probes.

Alternative probes (oligonucleotide, RNA, or biotinylated probes) may also be used. Contact GE Osmonics for technical assistance.

Clean probe solutions by adding a small amount of hybridization solution to the probe, and filter it through a Cameo 25AS supported cellulose acetate syringe filter (GE Osmonics catalog #DDA02025S0), to eliminate any contaminants before they come into contact with the transfer membrane.

Denature the probe by boiling in TE buffer for 5 minutes, or incubate with 0.1 volume of 1 N NaOH at 37°C for 5 minutes. Place on ice.

Add the decontaminated probe to the hybridization solution in the heat-sealable bag and reseal. Probe concentration should not exceed 20 ng/ml. Single copy genes or low copy message may require 1-5 10⁶ cpm/ml. Probes should be labeled no more than 24 hours before hybridization.

Hybridize 12 hours to overnight. Important: If the membrane is to be rehybridized, do not allow it to dry past this point. This will cause irreversible binding to the membrane.

Post Hybridization

Wash temperature should be 25°C below the Tm (melting temperature of the hybrid). If the homology between the probe and membrane-bound DNA is inexact, the wash temperature should be lower.

Stringency Washing Procedure

Low Stringency (for inexact matching)
2 x 15 minutes with 1 x SSC, 0.1% SDS at room temperature
2 x 15 minutes with 1 x SSC, 0.1% SDS at 37°C

Medium Stringency
2 x 15 minutes with 5 x SSC, 0.5% SDS at room temperature
2 x 15 minutes with 1 x SSC, 0.5-1.0% SDS at 37°C
1 x 15 minutes with 0.1 x SSC, 1.0% SDS at 37°C

High Stringency (for perfect hybrids)
2 x 15 minutes with 5 x SSC, 0.5% SDS at room temperature
2 x 15 minutes with 1 x SSC, 0.5-1.0% SDS at 37°C
3 x 15 minutes with 0.1 x SSC, 1.0% SDS at 65°C

NOTE: With nylon membranes, a medium or high stringency washing procedure is recommended to control potential background problems.

Use 0.5 ml of wash solution per square centimeter for all membranes. After washing, remove excess moisture with paper towels. Washes for Northern blots should substitute SSPE for SSC in all wash steps.

Autoradiography

Wrap the membrane in plastic wrap and autoradiograph at -70°C in a cassette with an intensifying screen while slightly damp. Expose the membrane for 25-60 hours.

Probe Removal

Do not allow the membrane to dry if a rehybridization step is intended. Wash in 5 mM Tris-HCl/pH 8.0, 0.2 mM EDTA, 0.05% pyrophosphate, 0.1 Denhardt's for 1-2 hours at 65°C. Rinse in 1 x SSPE. Or, wash in 50% formamide, 6 x SSPE at 65°C for 30 minutes. Rinse in 2 x SSPE.

Please call GE Osmonics' Labstore Support at (800) 444-8212, by fax (952) 988-6662 or email us for questions, pricing and availability. For international support and distributor locations, call +1-952-988-6665 or email us.