132
KAI-OLAF NETZER
The sensitivity of Southern blotting is sufficient to detect single-copy genes,
i.e. genes that exist in only one copy per genome. For medical applications,
the power of the method resides in the ability to analyze a small portion of
the primary structure of human genomic DNA.
Materials
• Agarosegel containing size fractionated DNA which has been digested to
completion with restriction enzymes prior to gel electrophoresis
• 20x SSC (3 M NaCl, 0.3 M sodium citrate)
• 0.25 M HCl
• Denaturing buffer (1.5 M NaCl, 0.5 M NaOH)
• Neutralization buffer (1.5 MNaCl, 0.5 M Tris-HCl, pH 7.5)
• Membrane filter: nitrocellulose filters, uncharged and charged nylon filters are available. Nylonfilters have better mechanical properties and are
therefore preferable. Charged nylon filters have slightly better DNA
binding capacity than uncharged nylon membranes, however, they
tend to give more background problems upon hybridization.
Procedure
ln advance
1. Digest genomic DNA samples (10 )lg) with the appropriate restriction
enzyme according to the principles given in Chap. 2.
2. Run an agarase gel as described in Chap. 4. U sually, 0. 7-1.0% agarase gels
are used to separate genomic DNA samples. They give a good resolution
over a size range of one to 15 kb. Either TBE or T AE buffer may be used.
Gels should be run at a valtage of 1 V/ern. Large gels (20 x 25 cm) should
be used if several bands of similar size are to be separated. Convenient
size markers are Hindill -digested Lambda DN A, Haeiii -digested DNA and 100 or 1000 bp size ladders. After electrophoresis, photograph
the gel on a UV transilluminator with a ruler alongside the gel. Take good
care not to drop or break the gel while moving it from the tray to the
illuminator.
Preparation of 1. Transfer the gel to a plastic box.
Southern blots
KAI-OLAF NETZER
The sensitivity of Southern blotting is sufficient to detect single-copy genes,
i.e. genes that exist in only one copy per genome. For medical applications,
the power of the method resides in the ability to analyze a small portion of
the primary structure of human genomic DNA.
Materials
• Agarosegel containing size fractionated DNA which has been digested to
completion with restriction enzymes prior to gel electrophoresis
• 20x SSC (3 M NaCl, 0.3 M sodium citrate)
• 0.25 M HCl
• Denaturing buffer (1.5 M NaCl, 0.5 M NaOH)
• Neutralization buffer (1.5 MNaCl, 0.5 M Tris-HCl, pH 7.5)
• Membrane filter: nitrocellulose filters, uncharged and charged nylon filters are available. Nylonfilters have better mechanical properties and are
therefore preferable. Charged nylon filters have slightly better DNA
binding capacity than uncharged nylon membranes, however, they
tend to give more background problems upon hybridization.
Procedure
ln advance
1. Digest genomic DNA samples (10 )lg) with the appropriate restriction
enzyme according to the principles given in Chap. 2.
2. Run an agarase gel as described in Chap. 4. U sually, 0. 7-1.0% agarase gels
are used to separate genomic DNA samples. They give a good resolution
over a size range of one to 15 kb. Either TBE or T AE buffer may be used.
Gels should be run at a valtage of 1 V/ern. Large gels (20 x 25 cm) should
be used if several bands of similar size are to be separated. Convenient
size markers are Hindill -digested Lambda DN A, Haeiii -digested DNA and 100 or 1000 bp size ladders. After electrophoresis, photograph
the gel on a UV transilluminator with a ruler alongside the gel. Take good
care not to drop or break the gel while moving it from the tray to the
illuminator.
Preparation of 1. Transfer the gel to a plastic box.
Southern blots