Preparation Protocol for FISH Probes

Fluorescence in situ hybridization (FISH) began with the combination of traditional cytogenetic and DNA techniques.
It is based on the Southern blot principle, where a known nucleic acid molecule indirectly labeled with a semi-antigen such as biotin, digoxin or directly labeled with fluorescein is used as a probe. The probe and target sequence double-stranded DNA is denatured and then hybridized. The complementary heterologous single-stranded DNA molecules are annealed at a suitable temperature and ionic strength to form stable heterologous double-stranded DNA, the semi-antigens are displayed by fluorescently labeled affinity or anti-digoxin antibodies, and the hybridization signal is observed by fluorescence microscopy.

The notch panning system for 1μg DNA is 0.1 mmol/L dTTP 6.5 μl, 0.1 mmol/L dNTP 10 μl, 10×Nick Translation buffer 5 μl, 1 mmol/L DIG-11-dUTP /Biotin-16-dUTP 0.5 μl, Nick Translation Enzyme 10 μl, DNA (1μg) X μl, and H₂O 18-X μl. For plasmids ≤10kb, react at 15°C for 3.5 hours; for BAC/PAC, react at 15°C for 9 hours.
Place the EP tube on ice, take 5 μl of it, and heat it at 70°C for 5 minutes, then perform 2% agarose gel electrophoresis to observe the size of the labeled probe, the suitable size of the single sequence probe is 200-600 bp; if the fragment size is large, extend the reaction at 15°C for 10-30 minutes, then repeat the gel electrophoresis until the suitable size of the probe is obtained.

Composition of the probe mixture. For BAC/PAC probes, DNA probe 5 μl, Human Cot-1 DNA 3 μl, Salmon Sperm DNA 0.5 μl, H₂O 1.5 μl. For mitotic probes, DNA probe 5 μl, Salmon Sperm DNA 0.5 μl, H₂O 4.5 μl.
Precipitation of DNA. The probe mixture was mixed with 1 μl (V/10) 3M sodium acetate (pH 5.2) and 27.5 μl (2.5V) anhydrous ethanol (lyophilized at -20°C), precipitated at -80°C for 30 min (or -20°C overnight), and centrifuged at 14,000 g at 4°C for 30 min to precipitate DNA.
Wash the precipitate. Carefully discard the supernatant, wash once with 70% ethanol, and centrifuge again at 14,000 g for 15 min at 4°C; carefully discard the supernatant and air-dry the DNA precipitate in a medium temperature water bath at 45-50°C for 10-15 min.
Dissolve the probe. Add 5 μl of pre-warmed deionized formamide (pH 7.0) at 37°C, centrifuge for a short time and shake at 37°C for 30 min to fully dissolve the DNA; then add 5 μl of pre-warmed Master Mix at 37°C, centrifuge for a short time and shake at 37°C for 15-30 min.
Denaturation and pre-hybridization of probes. Short centrifugation followed by denaturing probes in an 80°C water bath for 10 min and an ice bath for 5 min; short centrifugation and pre-hybridization in a 37°C water bath for 30-60 min; unique sequence probes (e.g. cDNA) and repeat sequence probes (e.g. α-satellite DNA) probes, no pre-hybridization is required.
Hybridize at 37°C overnight in a humidity chamber.

Creative Bioarray provides the most comprehensive list of FISH probes for the rapid identification of a wide range of chromosomal aberrations across the genome. We continue to expand the scope of the FISH probes to meet the customer's research needs- including the introduction of Customized FISH Probes.

When most of the ethanol evaporates, the white DNA precipitate becomes translucent; be sure to remove the ethanol thoroughly, otherwise, tiny precipitates will be generated after the addition of Master Mix, resulting in high background.
The DNA precipitate can also be dissolved in TE buffer 1.1 μl and diluted by adding Vysis probe buffer 4.4 μl, mixed and centrifuged instantaneously, and shaken for 15-30 min at 37°C.

For research use only. Not for any other purpose.