Hybridization parameters revisited: solutions containing SDS.

摘要: Salt concentration governs nucleic acid hybridization according to the Schildkraut-Lifson equation. High concentrations of SDS are used in some common protocols, but effects on stringency have not been reported. We investigated parameters solutions containing SDS. With targets immobilized nylon membranes and PCR- or transcription-generated probes, we report that 50% dissociation temperature (Tm*) absence was 15 degrees C-17degrees C lower than calculated Tm. had only modest Tm* [1% (w/v) equating 8 mM NaCl]. RNA/DNA hybrids were approximately 11 more stable DNA/DNA hybrids. Incomplete homology (69%) significantly reduced for (approximately /4degrees C; 0.45 C/% nonhomology) far less so 2.3 0.07 non-homology); incomplete also markedly extent hybridization. On these nylonfilters, a major effect nonspecific binding. Buffers lacking SDS, with low salt concentration, gave high backgrounds; buffers high-salt buffers, reproducibly backgrounds.