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0.2 mM SpectrumGreen, SpectrumOrangeTM or
Add 10 µL of 1 mM dUTP to 40 µ nuclease-free water.
0.1 mM dTTP
Add 10 µL of 0.3 mM dTTP to 20 µL nuclease-free water.
0.1 mM dNTP mix
Mix together 10 µL each of 0.3 mM dATP, 0.3 mM dCTP, and 0.3 mM dGTP.
Extracted P1, BAC, or YAC DNA
Prepare a 0.2µ g/µ to 1µ g/µ solution of extracted DNA in Tris-EDTA (10 mM Tris, 1 mM EDTA, pH 8.5) buffer.
This procedure labels approximately 1 µg of extracted P1, BAC, or YAC DNA. This is enough for ten FISH experiments (one target area equal to 22 mm x 22 mm).
Determining the Probe Size
Determining the probe size is an essential part of the labeling procedure. For detailed instruction on preparing and running an agarose gel see (1) Maniatis T, Fritsch EF, and Sambrook J. Gel electrophoresis of DNA. In: Molecular cloning: a laboratory manual. 2nd ed. Cold Spring, NY: ColdSpring Harbor Laboratory; 1989 or (2) Ausubel FM, ed. Preparation and Analysis of DNA. In: Current Protocols in Molecular Biology. New York: Greene Publishing Associates and John Wiley & Sons, 1989.
NOTE: Because of the isomeric structures of the fluorophores, the unincorporated dUTP appears on the agarose gel as two domains of differing brightness. The chemical structure and net charge of the different fluorophores affect the migration pattern on the gel. For example, unincorporated SpectrumGreenTM dUTP migrates to the top of the gel, while SpectrumOrangeTM and SpectrumRedTMdUTP migrate further down the gel and may overlap the probe DNA smear.In this case,you need to remove the unincorporated dUTP by ethanol precipitation and/or gel filtration of the labeled probe DNA before loading a sample of the probe DNA on the agarose gel for sizing.
As you increase the amount of enzyme and the incubation time, the size distribution shifts to progressively smaller probe fragments. To produce smaller probe fragments use the following conditions that are listed in order of decreasing fragment size:
5 µL enzyme mix/8 hour incubation, 5 µL enzyme mix/16 hour incubation, 10 µL enzyme mix/8 hour incubation and 10 µL enzyme mix/16 hour incubation. Adjust the amount of nuclease-free water added to keep the total reaction volumeat 50 µL.
The following is a recommended protocol for hybridization of unique sequence probes to lymphocyte cells. You may need to modify the procedure depending on the type and size of the probe.
Precipitating the Probe
Hybridizing the Probe to the Target Metaphase
Washing the Slide
Visualizing the Hybridization
Apply 10 µL of DAPI II counterstain and a coverslip to each hybridization location.
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