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0.2 mM SpectrumGreen or SpectrumRed dUTP
Add 10 µL of 1 mM dUTP to 40 µL 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 genomic DNA
Prepare a 0.2 µg/µL to 1 µg/µL solution of extracted genomic DNA in Tris-EDTA (10 mM Tris, 1 mM EDTA, pH 8.5) buffer.
This procedure labels approximately 1 µg of extracted genomic DNA. This is enough for five CGH hybridizations.
Determining the Probe Size
Determining the probe size is an essential part of the CGH 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: Molecularcloning: a laboratory manual. 2nd ed. Cold Spring, NY: Cold Spring Harbor Laboratory; 1989 or (2) Ausubel FM, ed. Preparation and Analysisof DNA. In: Current Protocols in Molecular Biology. New York: Greene Publishing Associates and John Wiley & Sons, 1989.
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 these conditions that are listed in order of decreasing fragment size: 5 µL enzyme mix/2 hour incubation, 5 µL enzyme mix/4 hour incubation, 10 µL enzyme mix/2 hour incubation and 10 µL enzyme mix/4 hour incubation. Adjust the amount of nuclease-free water added to keep the total reaction volume at 50 µL.
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