Thursday, March 19, 2009

Estimation of DNA Concentrations

The determination of the concentration of DNA or RNA in solution is a fundamental task in molecular biology. DNA is usually the limiting reagent in most experiments; therefore, the knowledge of its concentration is critical. Determination of the DNA concentration can be estimated either by qualitatively comparing the fluorescence of DNA bands in an agarose gel to a standard or by spectrophotometric means.

Qualitative Estimation

DNA fluorescence in the presence of ethidium bromide, and the intensity of the fluorescence is proportional to its concentration. As such, comparison of relative fluorescence of unknown DNA to known standards can be used as a rough estimation of DNA concentration. This comparison is usually made following the electrophoresis of the standard (for example, 1 microgram of Lambda DNA cleaved with the restriction endonuclease HindIII) and the unknown. The mass of 1 microgram of a Lambda-HindIII standard provides convenient references for comparison.

DNA concentration estimation

An alternative method for estimating DNA concentration relies on the lower limit for visually detecting DNA in a gel. It has been estimated that a band of DNA below 5 nanograms is not detectable by the human eye. Serially diluting DNA to extinction, followed by electrophoresis, can also be used to measure DNA concentration. This technique is particularly if there is more than one species or fragment of DNA in a sample. In this manner, the concentration of individual bands can be estimated.

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