Quantification and qualification of DNA/RNA/Protein

Introduction：

The quantification and purification test of the samples is the precondition, which helps you to decide, whether we can use them in downstream experiments.

The family of SHST's spectrophotometers helps you quickly assess, if your samples contain what you expect in appropriate amounts.

The core functions of SH-Nano series are the quantification and purity of the nucleic acids and protein, which are indispensable for the downstream experiments and applications.

Working Principles of a spectrophotometer:

Nucleic acids (incl. DNA and RNA)have the characteristic of ultraviolet absorption. The absorption wavelength of nucleic acids is 260 nm. According to Lambert Beer's theory, given the extinction coefficient of a certain substance and the thickness of the liquid layer, the concentration of the substance can be calculated due to its absorbance. Meanwhile, the purity of nucleic acids can also be estimated by calculating the ratio of A260/A280 and A260/A230. (The 280 nm absorption is usually from proteins, while 230 nm absorption is typically from sugars and phenols.)

Quantification:

The measurement that SH-Nano Series produce is the concentration of the sample in ng/uL. Since SH-Nano series provide ultraviolet absorbance, the quantification capabilities are inferior to the our Fluorometer (SH-FD100), which adopts the fluorimetric methods. Therefore, it is recommended to measure samples with more than 10 ng/uL of nucleic acids. (Click here to get more information of fluorometer SH-FD100)

Purity:

To deduce the purity of the samples is a very important purpose to use the spectrophotometer. This is generally indicated in two ratios: 260/280 and 260/230. These numbers correspond to the absorbance at the wavelengths 230, 260 and 280 nm.

The 260/280 ratio gives an indication of how pure the sample is from contaminating protein. Since proteins absorb at 280 nm, a low 260/280 ratio indicates the presence of high amounts of protein, relative to nucleic acids.

The optimal 260/280 ratio depends on the samples: RNA or DNA.

These values are as follow:

DNA: 1.80

RNA: 2.00

The 260/230 ratio is a value that reflects how pure the sample is from salts and other contaminants which can absorb at 230 nm.

Pure nucleic acid samples have a 260/230 ratio of 2 or above.

Anything less than 2 suggests that there are other factors in the sample.

Fluorometer (SH-FD100)

When shall we use Fluorometer?

The main preference for us to use a Fluorometer (SH-FD100) instead of a spectrophotometer (SH-NanoOne) is when the samples are with very low concentrations of nucleic acids. The sensitivity of our SH-FD100 can be as low as 10 pg/mL, which is superior to the SH-NanoOne.

The powerful SH-NanoOne PLUS

SH-NanoOne Plus provides both ultraviolet and fluorimetric methods.