Aging Aging

Differentially Expressed Genes 111Fig. 1. Northern blotting setup.3.10.2. Northern BlottingHere we describe a common method for Northern blotting that is performedwith the ordinary capillary flow system. As RNA is electrophoresedin the denaturing system the gel does not need any further denaturation orneutralization.The RNA is transferred from the gel onto a positively loaded nylon membranein a chamber construction shown in Fig. 1.The blotting setup is constructed in the following manner:1. Set up the transfer support into the tray and place a wider glass plate onto thesupport. Put two pieces of Whatman 3MM paper on the glass plate so that theyhang up into the tray. Fill the reservoir with transfer buffer (20× SSC) and wetalso the Whatman 3MM paper (avoid air bubbles).2. Cut a piece of the nylon membrane of the same size as the gel (handle the filter atthe edges only!) and wet the filter for 5 min in the transfer buffer. Cut off thelower right corner of the filter (so as not to lose orientation during the procedure).3. Flood the two Whatman 3MM papers on the glass plate with transfer buffer andplace the gel onto the papers. Squeeze out air bubbles by rolling with an RNasefreepipet over the gel. Cut a piece of the size of the gel out of the middle of apiece of common cling film and apply this around the gel. This will ensure thatthe liquid flow from the reservoir is transferred through the gel.4. Apply the nylon membrane carefully directly onto the gel and again squeeze outair bubbles as described previously.