How do I use FBM's borate media to resolve single-strand DNA in agarose and without chemical denaturants?

This application was published in BioTechniques in October, 2004. It takes advantage of the ability of low-salt solutions to retard the speed of DNA hybridization after melting has been accomplished. In our publication, we used a matched loading medium to melt the sample and to apply it to a well of a horizontal agarose submarine gel.

We made fresh a special 5X loading medium that contained 0.5X LB, 50% glycerol, and only trace orange G for the slightest coloration (the dye can be omitted to further reduce salt). 40 ng of each oligo were added to the loading medium and water to make a 1X sample containing 0.1X LB, 10% glycerol. The DNA of the sample was melted at 70 C for 5 minutes.

The melted sample was loaded onto a 3% agarose gel that had been pre-heated to 37 C in an incubator. Electrophoresis was done at 29 V/cm. Lower temperatures of gel did not work, presumably due to reduced stringency.

(Other methods of achieving sample melting would presumably be permitted, such as non- ... more.

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