By Walker J.M. (ed.)
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13. 14. 15 16 61 flotation on 2 X SSC, and place It on the gel. Smooth out an bubbles trapped between the gel and filter Drape strips of cling film from the edges of the gel to the edges of the tray. This prevents evaporatron of the SSC during transfer, and forces rt to move through the gel. Cover the nitrocellulose with two pieces of filter paper (20 x 20 cm) that have been wet m 2 x SSC. Divide a box of tissues or paper hand towels in two, and place over the filter paper. Put a glass plate on top of the tissues, followed by a 0 5-l kg weight.
This IS incubated with a 32P-labeled probe, which is DNA having a base sequence complementary to the DNA that IS to be detected on the filter After hybridrzation of the probe to Its complementary sequence, unbound probe is washed off. The posmon of the probe on the filter 1s then detected by autoradrography. This procedure was developed by E M. Southern of Edmburgh University (I), and is generally referred to as the Southern transfer or Southern blot. 55 56 Mathew The technique has two types of application.
The lower temperature limit (-30°C) is determmed by the vapor pressure of water. Colder temperatures take too long to dry the cells. 2 Water 1s used to resuspend cells if more salts from PBS would affect later biochemistry. Enough salts are contributed by the pellet to keep the cells from lysing when suspended m up to three pellet volumes of water lust before freezing. 3. The trypsm treatment of monolayer cultures has had no detectable effect on internal cell proteins and enzyme activities with thus rinsing scheme.