After hybridization the filters are washed several times under stringent conditions to remove the excess labeled probe. As explained in the last chapter on hybridization, the stringency of the buffer can easily be increased by lowering the molarity of the monovalent cations and/or increasing the temperature. For all the genomic sequencing experiments carried out in our laboratory, the washing buffers described in the ‘Step-by-Step Procedure’ were sufficient to give a low background. The molarity of sodium ion is kept at 74 mM for the more stringent washing buffer (washing buffer 2). The optimal temperature was determined empirically in the same way as described in the chapter III. 7.