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KAI-OLAF NETZER
Materials
• DNA (approximately 0.5 )lgl)ll in TE buffer)
• TE buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA)
• 20x SSC (3M NaCl, 0.3 M sodium citrate)
• Membrane filter: nitrocellulose filters, uncharged and charged nylon filters are available. Nylon filters 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 I. Sonicate genomic DNA at approximately 70 W for 1 minute on ice. Check
the fragment size by agarose gel electrophoresis. The fragments should
have an average size of 7 to10 kb.
Preparation
of dot blots
Note: DNA that has been digested with an appropriate restriction enzyme
may also be used. DNA should be purified by using a chaotropic buffer/silica-gel spin column system (e. g. Qiagen). Ifunfragmented genomic DNA is
used, hybridization may be less efficient.
2. Dilute the DNA to 0.5 )lgl)ll in TE buffer.
I. Heat 5 )lg DNA (in 10 )ll TE buffer) to 95°C for 10 minutes, then chill on
ice for 5 min. Collect the volume by brief centrifugation in a microfuge.
Place tube on ice.
2. Cut a piece of filter membrane to the appropriate size. Mark the locations
where the DNA will be spotted with a soft pencil. A one cm-grid may be
helpful.
3. Prewet the membrane briefly in distilled water, then saturate it in 20x
SSC. Place the membrane on a filter paper wad (Whatman 3MM)
pre-saturated in 20x SSC.
4. Spot the DNA samples on to the filter in 5 )ll aliquots. Allow each spot to
dry before applying the second aliquot.
Note: The filter should remain damp but not too wet throughout the spotting of DNA samples.
KAI-OLAF NETZER
Materials
• DNA (approximately 0.5 )lgl)ll in TE buffer)
• TE buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA)
• 20x SSC (3M NaCl, 0.3 M sodium citrate)
• Membrane filter: nitrocellulose filters, uncharged and charged nylon filters are available. Nylon filters 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 I. Sonicate genomic DNA at approximately 70 W for 1 minute on ice. Check
the fragment size by agarose gel electrophoresis. The fragments should
have an average size of 7 to10 kb.
Preparation
of dot blots
Note: DNA that has been digested with an appropriate restriction enzyme
may also be used. DNA should be purified by using a chaotropic buffer/silica-gel spin column system (e. g. Qiagen). Ifunfragmented genomic DNA is
used, hybridization may be less efficient.
2. Dilute the DNA to 0.5 )lgl)ll in TE buffer.
I. Heat 5 )lg DNA (in 10 )ll TE buffer) to 95°C for 10 minutes, then chill on
ice for 5 min. Collect the volume by brief centrifugation in a microfuge.
Place tube on ice.
2. Cut a piece of filter membrane to the appropriate size. Mark the locations
where the DNA will be spotted with a soft pencil. A one cm-grid may be
helpful.
3. Prewet the membrane briefly in distilled water, then saturate it in 20x
SSC. Place the membrane on a filter paper wad (Whatman 3MM)
pre-saturated in 20x SSC.
4. Spot the DNA samples on to the filter in 5 )ll aliquots. Allow each spot to
dry before applying the second aliquot.
Note: The filter should remain damp but not too wet throughout the spotting of DNA samples.