DNA
Quantification and normalization of DNA is traditionally done by measuring the absorbance at 260nm (A260). However, for impure DNA samples, the A260 read-out can lead to an overestimation of the DNA concentration due to UV-absorbing contaminants.
Alternative methods, such as the Quant-iT™ PicoGreen® assay, based on fluorescence enhancement upon binding with dsDNA, have been developed to address this issue. While offering improved specificity and sensitivity, such assays are less convenient, requiring multiple pipetting steps and running additional standards.
The A260/A280 and A260/A230 ratios are commonly used to quickly assess the purity of samples. Unfortunately those ratios are insensitive purity indicators and not reliable enough (See example below). Components as phenol or guanidinium salts used in extraction buffers can be copurified with the DNA. Such contaminants can drastically affect the success and accuracy of downstream applications.
The DropSense96 platform combined with cDrop software provides a tool for both specific dsDNA quantification and purity evaluation by determining the contaminations present in the sample.
For more details on how the DropSense96 and the cDrop software can help you regarding your main application, please see other sections in the left menu.
This technology is ideal to help with:
- Specific quantification of DNA
- QC of DNA samples prior storage, NGS, qPCR…
Lack of sensivity of Ratios to assess sample purity
Samples were prepared by mixing a dsDNA sample with varying amounts of RNA, Protein and Phenol. DNA was measuring with the A260 method leading to an overestimation of the DNA concentration due to the presence of these contaminants. Moreover no relevant information on the purity can be provided as the presence of proteins or phenol do not modify significantly the A260/A280 and A260/A230 ratios.
A cDrop analysis was also performed on same samples to obtain a specific and accurate concentration of dsDNA.
Moreover the presence of other components is also quantified giving an accurate overview of the purity of each sample. Such information can greatly help to eliminate outliers or to improve some experimental steps as for instance the extraction process.
