Non-denaturating Nucleic Acid Electrophoresis

This system separates double-stranded DNA (dsDNA) fragments with a length of 70 x 80 000 base pairs (bp) in gels of 3% to 0.1% agarose. For analysis of smaller-fragments (6000 to 1000 bp) PAGE systems are described in literature with 20-3% polyacrylamid. 

During sample preparation and electrophoresis DNA will not be denaturated, i.e., the double strand and higher elements of structure stay unaffected. To avoid unwanted further fragmentation all manipulations have to be done without possible contaminations by fingerprints, droplets of saliva or microorganisms. Sterilized materials are recommended. 

Since protocols for electrophoresis used in DNA sequence analysis are given, for example, by Sambrock et al., these specialized methods are not outlined in this chapter. 

Caution Wear gloves Ethidium bromide is carcinogenic. 

The agarose (Soln. B) is molten in aboiling water bath and cooled to about 50 °C. Soln. D is added to a final concentration of 0.5 pg/mT . The liquid agarose is poured into the gel chamber of a horizontal electrophoresis apparatus and the comb is inserted. 

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