of salt increasing the concentration of formamide or raising the hybridization temperature for example high stringency conditions could occur at about 50 formamide at 37 u00b0 c to 42 u00b0 c reduced stringency conditions could occur at about 35 to 25 formamide at 30 u00b0 c to 35 u00b0 c examples of stringency conditions for hybridization are provided in sambrook j 1989 molecular cloning a laboratory manual cold spring harbor laboratory press cold spring harbor n y further examples of stringent hybridization conditions include 400 mm nacl 40 mm pipes ph 6 4 1 mm edta 50 u00b0 c or 70 u00b0 c for 12 16 hours followed by washing or hybridization at 70 u00b0 c in 1 u00d7 ssc or 50 u00b0 c in 1 u00d7 ssc 50 formamide followed by washing at 70 u00b0 c in 0 3 u00d7 ssc or hybridization at 70 u00b0 c in 4 u00d7 ssc or 50 u00b0 c in 4 u00d7 ssc 50 formamide followed by washing at 67 u00b0 c in 1 u00d7 ssc the temperature for hybridization is about 5 10 u00b0 c less than the melting temperature t m of the hybrid where t m is determined for hybrids between 19 and 49 base pairs in length using the following calculation t m u00b0 c 81 5 16 6 log 10 na 0 41 g c u2212 600 n where n is the number of bases in the hybrid and na is the concentration of sodium ions in the hybridization buffer the above described in vitro hybridization assay provides a method of predicting whether binding between a candidate sirna and a target will have specificity however in the context of the risc complex specific cleavage of a target can also occur with an.