ELECTROPHORETIC SEPARATION OF NUCLEIC ACID COMPONENTS 275 



5 

 (cm.) 



10 



Fig. 3. The electrophoresis apparatus." 



stances are loeated by making a contact print in monochromatic ultra- 

 violet light of 260 m/i,^*^' *^ and may be observed during the run by the 

 technique of Holiday and Johnson.-" 



The carbon tetrachloride keeps the paper cool and prevents evaporation. 

 It was chosen for three important reasons: (1) it is reasonably transparent 

 to ultraviolet light; (2) it is nonpolar and so has no tendency to dissolve 

 out the substances from the paper; (3) it is denser than water and so pre- 

 vents buffer from siphoning over from the electrode vessels and water- 

 logging the paper. 



With Whatman No. 3 mm. paper about 2 mg. substances may be easily 

 run per centimeter width of paper; the only effect of increasing the amounts 

 put on is to make the separating bands broader. Placing the substances in a 

 line rather than in circular spots gives a much sharper separation. 



After an electrophoretic run the substances often have to be submitted 

 to further chromatographic analyses. This is greatly facilitated if the buf- 

 fer components move as spots on the chromatograms, or if they are vol- 

 atile; for this reason buffers such as ammonium formate and ammonium 

 acetate are the most suitable. Using 0.05 M ammonium formate or acetate 

 buffer in the size of apparatus described above, at 20 v. /cm. the current 

 is about 8-10 ma. However, with the 0.05 M phosphate and borate buffers 

 used for the ranges pH 6-8 and 8-10, it is often necessary to reduce the 

 voltage gradient to about 15 v. /cm. to prevent overheating. 



18 R. Markham and J. D. Smith, Biochem. J. 45, 294 (1949). 

 " R. Markham and J. D. Smith, Biochem. J. 49, 401 (1951). 

 =*» E. R. Holiday and E. A. Johnson, Nature 163, 216 (1949). 



