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HARRY SCHERTHAN
Procedure
Probe labeling
Nick translation, random priming and PCR protocols have proven to be
effective methods for labeling probe DNAs. For ease of handling, commercially available labeling kits may be utilized. For a detailed description of
nick translation, the reader is referred to step by step protocols published
elsewhere (e.g., Cremer and Lichter 1992). A critical point in Zoo-FISH is
the fragment size and concentration of the labeled DNA probe. Fragment
sizes of 100-300 bp have yielded good signal-to-noise ratios. It is recommended to monitor the size and amount of labeled probe by running an
aliquot e1 20 ) of the nick reaction on a 1% agarose mini gel.
1. Obtain purified WCP DNA probe by plasmid preparation or PCR.
Make sure that the probe DNA is free of RNA.
2. Labell Jlg of pBS chromosome specific DNA library probe by nick
translation using a commercial kit according to the recommendations
of the supplier (e.g. BioNick Kit; Life Technologies).
3. After the appropriate incubation time, set 1 JlI of each reaction aside
and perform minigel analysis and a dot blot test to control the amount
and quality of DNA labeling, respectively (Scherthan and Cremer
1994).
4. Precipitate labeled DNA probe (1 Jlg) by addition of3 Jlg of human Cotl
DNA and 3 vol of ethanol.
5. After incubation at -20°C for 30 min overnight, spin for 30 min at high
speed.
6. Discard supernatant and wash pellet briefly with 70% ethanol.
7. Air dry pellet by placing the open tube for 5-10 min in a dry incubator
or drying block at 65°C.
8. When remnants of liquid have evaporated, dissolve pellet in 3.3 JlI hybridization solution.
HARRY SCHERTHAN
Procedure
Probe labeling
Nick translation, random priming and PCR protocols have proven to be
effective methods for labeling probe DNAs. For ease of handling, commercially available labeling kits may be utilized. For a detailed description of
nick translation, the reader is referred to step by step protocols published
elsewhere (e.g., Cremer and Lichter 1992). A critical point in Zoo-FISH is
the fragment size and concentration of the labeled DNA probe. Fragment
sizes of 100-300 bp have yielded good signal-to-noise ratios. It is recommended to monitor the size and amount of labeled probe by running an
aliquot e1 20 ) of the nick reaction on a 1% agarose mini gel.
1. Obtain purified WCP DNA probe by plasmid preparation or PCR.
Make sure that the probe DNA is free of RNA.
2. Labell Jlg of pBS chromosome specific DNA library probe by nick
translation using a commercial kit according to the recommendations
of the supplier (e.g. BioNick Kit; Life Technologies).
3. After the appropriate incubation time, set 1 JlI of each reaction aside
and perform minigel analysis and a dot blot test to control the amount
and quality of DNA labeling, respectively (Scherthan and Cremer
1994).
4. Precipitate labeled DNA probe (1 Jlg) by addition of3 Jlg of human Cotl
DNA and 3 vol of ethanol.
5. After incubation at -20°C for 30 min overnight, spin for 30 min at high
speed.
6. Discard supernatant and wash pellet briefly with 70% ethanol.
7. Air dry pellet by placing the open tube for 5-10 min in a dry incubator
or drying block at 65°C.
8. When remnants of liquid have evaporated, dissolve pellet in 3.3 JlI hybridization solution.