Increased 32P-SSCP sensitivity by combining RE digestion and extended X-ray film exposures.

Low-power vacuum pumps are commonly used for Southern and Northern transfer from agarose gels and for slot and dot blotting. Unfortunately, commercially available vacuum pumps are expensive and susceptible to contamination with transfer reagents. The following describes the use and construction of a very simple and cheap apparatus, which generates and measures a partial vacuum as effectively as commercially available pumps (Figure 1). It functions on the principle that water attempting to move out of the drum will generate a partial vacuum proportional to the difference in height between the upper water level and the outlet (e.g., 40 cm). See Table 1 for use. At this stage, approximately 100 mL of water escape from “straight connector-a” for about 5–10 s, but should then essentially stop. Continued loss of water indicates a leak. The water levels in the two arms of the vacuum gauge should now have moved to the points marked “v”, where the distance between them gives the vacuum obtained (e.g., 40 cm). Thus, the vacuum is determined by adjusting the height of “straight connector-a”. During transfer of 2 h, 100–200 mL escape from “straight connector-a”. Thus, the use of a large-diameter drum results in only a small drop in water level (200 mL result in about a 3–4mm drop). For construction of the vacuum pump, the Plasticware must consist of: (i) an aspirator bottle (25 L), (ii) a T untapered connector (3.3-mm valley, 3.6mm crest), (iii) a straight connector tapered (a) (10–12-mm outside diameter), (iv) a straight connector tapered (b) (4–6-mm outside diameter) and (v) tubing measurements for inside diameters. To put “straight connector-b” into the drum, just drill a hole and wrap Parafilm® around the connector. It is handy to have “straight connector-a” pointed slightly upwards, to stop air bubbles from entering the tubing.