Spectrophotometry is a mature technique for the quantification and analysis of biological components. Among them, nucleic acid is one of the most commonly detected biological components in biological laboratory. Determining the concentration and purity of these samples is essential for many downstream experiments.

Nucleic acids mainly absorb ultraviolet light at 260nm, and the concentration can be calculated by using Lambert-Beer's law from their relative extinction coefficients and sample optical path. First, ultraviolet light at 260nm shines directly on the sample and passes through it, while a photodetector on the other side measures how much light is absorbed. The nucleic acid concentration in the sample can be quantified by a reference (usually a sample diluent).

Sample purity is an important index of nucleotide quantification. Although not the most accurate method for determining purity, A260/A280 and A260/A230 can still be used to estimate the degree of contamination of protein and chemical composition.

The Ultra-Micro Spectrophotometer is a multi-purpose Ultra-Visibility (UV-Vis) Ultra-Micro Spectrophotometer, especially for the analysis of nucleic acid samples.

In this application guide, we show how an ultramicroscopic UV spectrophotometer can be used to analyze nucleic acid samples quantitatively (concentration) and qualitatively (sample purity) with high accuracy and consistency.

Let's walk into a few experiments in our lab.

(1) Sample cross contamination:

Procedure: Sample cross-contamination was evaluated by alternate detection of salmon extract (dsDNA) and fetal bovine serum albumin (BSA) using our NanoBio200 ultramicroscopic spectrophotometer. Ultra pure water as a reference. The ultra-micro base is wiped clean with non-fuzz paper after each reading.

The experimental results are as follows:

Figure 1: Sample cross contamination. Salmon extract (dsDNA) and fetal bovine serum albumin (BSA) were detected alternatively on NanoBio200 ultramicroscopic spectrophotometer.

Table 1: Data contained in the experiment of "sample cross-contamination" From the results in Figure 1 and Table 1, it can be seen that there was no obvious sample cross-contamination in the subsequent experiments after simple wiping of the mirror wipe paper.

(2) Sample volume comparison:

Experimental procedure: Salmon extract (dsDNA) was diluted by ultra TE with 1.0μL, 2.0μL and 2.5 μL, respectively, and detected in ultrafine droplets (n = 5). Te as a reference. The reference volume is the same as the sample volume.

Experimental results:

Figure 2: Volume reproducibility. Salmon extract (dsDNA) in volumes of 1ul, 2ul and 2.5ul were read on NanoBio200 ultramicroscopic spectrophotometer.

Table 2: Comparison data of different volumes

From Figure 2 and Table 2, we can see that different volumes show very consistent calculated concentrations (n = 5), indicating that the amount of our volume has little effect on the concentration determination of the sample, as long as a perfect liquid column is formed. To get the best results, we recommend a 2ul volume as it is easier to sample.

(3) Linearity of standard curve:

The process of the experiment was as follows: DNA of salmon extract was dissolved in double stranded DNA(dsDNA) in TE buffer solution (pH =8.0) with a starting concentration of 2000ng/μL and then diluted by double series. Te was used as blank, and the concentration of each sample was read 3 times on the 0.5mm ultramicrometric base. The dsDNA concentration was automatically calculated using the preset DNA quantitative method, and cdsDNA =50[A260(10mM)-A340(10mM)]ng/ul. After that, the data can be charted using Excel imports and curve fitting is used to show the linearity of the standard curve.

Experimental results:

Figure 3: Calculated DNA concentration vs dilution factor. The relationship between dilution factor and DNA concentration of Salmon Extrine DNA at a starting concentration of 2154ng/ μL was shown by a series of double dilutions obtained from Nanobio200. This curve shows a perfect linear relationship with an R2 value of 0.9991.

From Figure 3, we can see that NanoBio200 shows a linear relationship between DNA concentration and dilution factor. From this experiment, we can get the minimum detection concentration of this NanoBio200 is 5ng/μL DNA.

Conclusion:

NanoBio200 ultramicroscopic spectrophotometer can be used for very sensitive nucleic acid sample quantification and analysis. As shown above, samples as low as 1 μL can yield very consistent readings in ultramicroscopes. It also has a very wide detection range (5 ng/μL to 2154 ng/μL).

Also, its 7-inch capacitive touch screen provides important information for researchers.

From Figure 4, we can intuitively see the sample concentration and purity. Small and flexible volume, plus it does not need a computer online, a single machine can be detected, the detection data can be printed, but also through USB and other ways to output advantages. This makes the NanoBio200 ultra-micro spectrophotometer an ideal choice for nucleic acid quantification and analysis in any laboratory environment.