Posthybridization washes/stringency

Posthybridization entails a series of washes with preset stringency conditions to adjust the specificity or the wanted degree of homology and to wash out the unbound probe. Hence, posthybridization washes ordinarily include specified amounts of formamide and sodium chloride and sodium citrate (SSC) stock to adjust for the degree of stringency. 

Inclusion of formamide in the HM mix and posthybridization washes lowers the melting temperature of DNA. Denaturation, hybridization, and stringency washes in formamide, therefore, helps preserve the architecture of the chromosomes, by lowering the denaturation and stringency washing temperature. 

Five steps can be distinguished in membrane hybridization (i) immobilization of target nucleic acid (ii) prehybridization to saturate the remaining binding sites which would otherwise adsorb probe non-specifically (iii) hybridization in low stringent conditions to adsorb probe as efficiently as possible (iv) posthybridization washes to define the stringency of the hybridization and thus the specificity of the reaction (v) the detection step (Fig. 8.2). In addition, the hybridized probes can sometimes be stripped from the blots to expose the targets to other probes. 

---