180
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
• Single cosmids, phages, plasmids: 40-80 ng
• Isolated inserts derived from individual sorted human chromosomes:
100-300 ng
• Gel-purified or PCR amplified single YACs: 50-150 ng
Materials
• Sheared salmon sperm DNA
• Cotl-DNA fraction ofhuman genomic DNA at a size of approximately
500nt
• Salutions and labeled probe as described in Subprotocols 4 and 6
Procedure
Hybridization 1. Combine the appropriate amount oflabeled probe DNA (see above), 2-4
Jlg of human competitor DNA (Cotl-fraction) and add salmon sperm
DNA to a total of 10 Jlg DNA.
2. Precipitate DNA by adding 1/20 volume of sodium acetate, pH 5.2 and 2
volumes of ethanol. Incubate at -70°C for 30 minutes. After centrifugation at 12,000 rpm for 10 minutes, in most cases a pellet will be visible.
Discard supernatant and wash with 70% ethanol. Re-centrifuge and discard supernatant.
3. Lyophilize sample and resuspend the pellet in 5J.!l of deionized formamide (see Subprotocol 4).
4. Add 5 J.!l of denaturation buffer.
5. Denature the DNA by incubation at 75°C for 5 minutes.
6. Quickly transfer the microcentrifuge tube containing the probe solution
to 37°C. Incubate for 5-20 minutes (or even longer) to allow partial reannealing.
7. Apply pre-anneled probe mixture to the denatured DNA on the slides.
8. Continue as described in Subprotocols 4, 5 and 6.
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
• Single cosmids, phages, plasmids: 40-80 ng
• Isolated inserts derived from individual sorted human chromosomes:
100-300 ng
• Gel-purified or PCR amplified single YACs: 50-150 ng
Materials
• Sheared salmon sperm DNA
• Cotl-DNA fraction ofhuman genomic DNA at a size of approximately
500nt
• Salutions and labeled probe as described in Subprotocols 4 and 6
Procedure
Hybridization 1. Combine the appropriate amount oflabeled probe DNA (see above), 2-4
Jlg of human competitor DNA (Cotl-fraction) and add salmon sperm
DNA to a total of 10 Jlg DNA.
2. Precipitate DNA by adding 1/20 volume of sodium acetate, pH 5.2 and 2
volumes of ethanol. Incubate at -70°C for 30 minutes. After centrifugation at 12,000 rpm for 10 minutes, in most cases a pellet will be visible.
Discard supernatant and wash with 70% ethanol. Re-centrifuge and discard supernatant.
3. Lyophilize sample and resuspend the pellet in 5J.!l of deionized formamide (see Subprotocol 4).
4. Add 5 J.!l of denaturation buffer.
5. Denature the DNA by incubation at 75°C for 5 minutes.
6. Quickly transfer the microcentrifuge tube containing the probe solution
to 37°C. Incubate for 5-20 minutes (or even longer) to allow partial reannealing.
7. Apply pre-anneled probe mixture to the denatured DNA on the slides.
8. Continue as described in Subprotocols 4, 5 and 6.