@The concentration of DNA or RNA in a sample is most accuratly determined by absorbance at 260nm. More often, the sample is too small or too dilute for this method, which generally requires ug amounts of the sample for accurate measurement. A fast and relatively accurate method for RNA/DNA concentration estimation is the EtBr spot test. This method will usually allow the estimation of concentration using only ng amounts of sample.
- Materials
- 0.1ug/ml standards
Dissolve lambda DNA or E.coli rRNA in TE to ~1ug/ml, then accurately determine the concentration by A260 (an A260 of 1 = 50ug DNA/ml or 40ug RNA/ml). Dilute to 100ng/ml in TE, & preparae dilutions of 90, 80, 70, 60, 50, 40, 30, 20, and 10ng/ml from this stock.
- Methods
- On a petri dish or glass slide, spot 10ul aliquots of 10, 20, 30, 40, 60, 80 and 100ng/ml ribosomal RNA or lambda DNA (depending on whether your sample is RNA or DNA).
- Prepare serial 1:2 dilutions of your sample (usually 5-8 are enough) in TE, and spot 10ul of each of hese dilutions onto the petri plate.
- To each spot, add 10ul 0.2ug/ml ethidium bromide, & mix by pipetting up & down.
- Using a transilluminator or hand-held UV source, photograph the plate.
- Estimate the concetration of the sample by conparison of the dilutions of the samples with each of the standard DNA or RNA samples.