274 J. D. SMITH 



n. Apparatus and Techniques 



The first attempt at the separation of nucleic acid components by electro- 

 phoresis was made by Gordon and Reichard,^^ who ran deoxyribonuclease 

 digests of deoxyribonucleic acid on agar gel. The use of agar as a supporting 

 medium has obvious disadvantages. It is difficult to avoid overheating and 

 consequent "sweating" of the gel, and the recovery of the separated ma- 

 terial from the agar is cumbersome. 



Paper is a much more suitable supporting material both for analytical 

 and preparative work. The chief problems in paper electrophoresis lie in 

 avoiding the siphoning of buffer down the paper from the electrode vessels, 

 and the evaporation of water from the paper due to heating during the 

 run. Both of these prevent the maintenance of a uniform concentration of 

 bufTer along the paper. Two types of apparatus originally designed for the 

 separation of amino acids have been used with nucleic acid derivatives. 

 One is the apparatus described by Durrum^* in which the paper strip passes 

 over a glass support and both ends dip into buffer solution, the whole being 

 enclosed in a glass vessel to minimize evaporation. In the second method 

 the paper is held between two glass plates which are cooled by circulating 

 liquid. ^^ Neither of these methods satisfactorily overcomes the problem of 

 evaporation from the paper, and both have to be used with comparatively 

 low voltage gradients. 



A more convenient technique has been described by Markham and 

 Smith^' " where the paper is kept cool by immersion in carbon tetrachloride. 

 The apparatus (Figure 3) consists of three rectangular glass jars A, B, 

 and C (convenient sizes for most work are A, and B, 15 x 10 x 5 cm., C, 

 21 X 12 X 6.5 cm.). A and B contain the buffer solution and C contains car- 

 bon tetrachloride. A strip of Whatman No. 3 mm. paper, 56 x 8 cm., is 

 soaked in the buffer solution, blotted to remove surplus moisture, and 

 about 0.1-0.2 ml. of a solution containing the mixture to be separated ap- 

 plied in a line across the paper at a suitable place, usually about 12 cm. 

 from one end. The paper is placed in the apparatus so that the two ends 

 dip into vessels A and B and the center part of the paper is immersed in 

 the carbon tetrachloride in vessel C, being held in place by a piece of cel- 

 luloid or glass (celluloid often contains ultraviolet-absorbing substances 

 soluble in CCI4). The electrodes, two arc carbons, are placed in A and B 

 and the whole covered by a glass plate. The electrodes are connected to a 

 d.c. power supply, usually of 1000 v. thus giving a voltage gradient of 

 about 20 v./cm. (Suitable powder supplies are shown in Figure 4.) Under 

 these conditions a 2-hr. run is adequate for ost eparations. The sub- 

 's E. L. Durrum, J. Am. Chem. Soc. 72, 2943 (1950). 

 !» T. Wieland and E. Fischer, Naturwissenchaften 35, 29 (1948). 

 17 R. Markham and J. D. Smith, Biochem. J. 52, 552 (1952). 



