Sample Prep for Denaturing PAGE of DNA

DNA samples for denaturing gel electrophoresis must be denatured prior to loading, to avoid time dependent denaturation artifacts on the gel. This is usually carried out by diluting the sample into 95% formamide and heating to 95°C (see the Dideoxy Sequencing (Taq Polymerase) Protocol for a formula for the loading buffer).

Loading the proper amount of DNA is critical for good results. Too little DNA will not be detected, while overloading lanes leads to smearing of bands. Acrylamide gels have a relatively high sample capacity—up to 10 µg can be loaded per lane in many cases.

Determining sample concentration

The concentration of DNA in the sample may be determined in several ways. The most straightforward is to make use of the absorbance at 260nm of the nucleotide bases. Pure DNA at a concentration of 50 µg/ml has an A260 of 1.0 (Concentration is linear with absorbance by Beer's Law). The purity of the DNA may be checked at the same time: pure DNA has an A260/ A280 ratio of 1.8. A lower ratio indicates protein contamination; a higher ratio indicates substantial RNA content. Lower concentrations of DNA may be assayed by taking advantage of the fact that the fluorescence of DNA/ethidium bromide complexes is proportional to the concentration of DNA in the sample. Levels of DNA as low as 10 ng can be quantified in a 10ml volume by diluting the DNA into buffer or water containing 0.5 µg/ml Ethidium Bromide. CAUTION: ETHIDIUM BROMIDE IS A POWERFUL MUTAGEN. Comparing the fluorescence of serial dilutions of sample with the fluorescence of known standards allows the determination of the DNA concentration in the original sample.

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