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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 a ratio of A260/ A280 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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Products Related to this Discussion:
Bromophenol Blue
Tracking dye that comigrates with smaller macromolecules through PAGE and Agarose gels.
Formamide - ULTRA PURE
Deionized and packed under nitrogen. Ready-to-use. Electrophoresis grade.