Wash stringencies

The variables that must be considered are the same as those considered for standard in-situ hybridization of embryos Tanperature, probe concentration, and washing stringency must all be optimized. For some probes, better results are obtained if the explants are removed from either one half or all of the collagen after fixation and processed in small vials. 

The hybridization and washing conditions for library screening should be carried out at a lower stringency, particularly when the probe and library are derived from different species. In the example in Fig. 1, a mouse was used to screen a human library, and washing stringency was kept low (45°C and 2x SSC). 

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. 

Extremely stringent wash conditions. (Oligoprobes can be washed away under very high stringency.)... 

Unfortunately, the conditions for the immunoprecipitations (especially the stringency of the binding and wash buffers) have to be determined experimentally for each antibody. These stringent conditions, on the one hand, can help to reduce the background, but on the other hand, can result in a loss of signal. 

Use for stringency wash steps after the hybridization step. 

Wash m the appropriate solution for stringency, e g, 50% formamide/O. IX SSC, if required for discriminating closely homologous sequences (see refs 11-13) Adjust all washing solutions to pH 7.0 with 5M HC1 The temperature of the solution should be monitored directly using a mercury thermometer. Washing should be carried out for 30 min... 

Inclusion of formamide is essential for lowering the melting temperature of DNA. It is necessary to perform denaturation and hybridization of probe and chromosomal DNA at a lower temperature to ensure preservation of chromosomal architecture. Similarly, inclusion of formamide in the stringency washes increases the stringency without having to increase the temperature... 

Reducing the salt concentration (from 2X SSC to 0.1 X SSC) during washing also increases the stringency. 

To prevent the selection of aptamers with low affinity to the target molecule, the target concentration has to be decreased and the washing procedure has to be done with increased stringency. This enhances the discrimination between aptamers with high affinity and aptamers with lower affinity [6]. 

The eluted pool is a heterogeneous population which contains phages that bind with different affinities as well as phages adsorbed aspecifically, that are not removed even through extensive washing steps. As a consequence, in order to isolate the binding peptides, it is necessary to increase the stringency of selection. 

Because of the sequence complexity of genomic DNA, isolation of false positive clones by blot hybridization is a significant problem when the stringency of the washing conditions is very low (wash temperature <42°C). 

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