Mouse Cot-1 DNA

For Southern blot hybridizations, add 50 µg of COT-1 DNA (at 10 µg/µL) to 50 µL of 20X SSC, 25 µL distilled water and 20 µL of a solution containing 0.1 M NaCl, 0.1 M Tris-HCl (pH 7.4). 0.01 M EDTA, and 1% SDS to the probe for each 25 to 500 ng of probe. For in situ hybridizations, combine genomic probe with the proper amount of COT-1 DNA such that the final concentration of COT-1 DNA is 0.3 µg/µL for cosmid, plasmid, and lambda probes; or, at 1 µg/µL for Alu PCR probes. Ethanol precipitate and resuspend in a half-volume of 100% formamide. Add a half-volume of 20% dextran sulfate in 2X SSC (prewarmed to 75 degrees C) and mix well. Denature mix by heating to 75 degrees C for 5 min. Incubate at 37 degrees C for 5 to 15 min.

Probes can be labeled with 32P by random primer or nick translation procedures using the Random Primers DNA Labeling System (Cat. No.18187-013) or Nick Translation System (Cat. No. 18160-010). Biotinylated COT-1 DNA can be prepared by nick translation with the BioNick Labeling System (Cat. No. 18247-015) or by the BioPrime DNA Labeling System (Cat. No. 18094-011). Improved results can be obtained when the COT-1 DNA is first ligated to itself to provide an optimum template.