Human Cot-1 DNA™-Fluorometric QC

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.

Human Cot-DNA-Fluorometric QC has been quantitated by fluorometry; this provides more accurate concentration measurements for higher molecular weight products. Mouse Cot-1 DNA blocking reagent is recommended for experiments when blocking of repetitive mouse DNA is required.

Cot-1 DNA blocking reagent blocks repetitive sequences such as SINEs (short interspersed elements), LINEs (long interspersed elements), and sequence homology among members of the same gene family when added to the hybridization solution.

Better results are obtained when Cot-1 DNA blocking reagent is used due to reduction in cross hybridization, which leads to cleaner, more sensitive aCGH microarray experiments and FISH assays.