Measurement Reproducibility with the Genova

By AZoM.com Staff Writers

Table of Content

Introduction
Methods
Results Colorimetric samples
dsDNA
Protein
Tips for Obtaining Reproducible Results
Conclusions
About Bibby Scientific

Introduction

When micro volume samples are measured, minute changes in absorbance may result in greater differences in the concentration values calculated because of the inherent "dilution" factor of the small path length. Hence it is very important that the micro volume spectrophotometer used provides repeatable and stable absorbance readings. This article reports measurement of a number of different sample types including nucleic acids, protein and food coloring. The Jenway Genova Nano is used to demonstrate both stability in absorbance and reproducibility in derived sample concentration.

Methods

The Genova Nano was setup in the appropriate mode for each experiment. 2µl of nuclease-free water was used to zero the instrument and then 2µl aliquots of the various solutions were measured. For each sample, 10 consecutive aliquots were measured and the sample was wiped off the read head each time with a lint-free cloth. All the measurements were performed with the optional background correction on.

The samples used included a solution of green food colouring, a sample of egg white protein, calf thymus DNA (Sigma D3664), samples of yeast RNA (Sigma R6750) and an oligonucleotide PCR primer. The samples were diluted as needed with nuclease-free water to obtain the required concentration ranges.

Results Colorimetric samples

A sample of green food coloring (Supercook, Leeds, UK) was used in order to measure absorbance repeatability. This includes a mixture of tartrazine(E102) and Green S(E142). Figure 1 shows the absorbance peak of the food coloring showing a broad peak at around 429nm and a sharper peak at around 640nm, hence these wavelengths were selected for measurement. The food coloring was diluted 1 in 10, 1 in 50 and 1 in 100 in water.

Figure 1. Visible absorbance spectrum of green food colouring measured using the micro volume accessory at 0.5mm path length showing peaks at 428nm and 640nm.

The Genova Nano was set up in multiwavelength mode to measure at both wavelengths. Table 1 provides the absorbance values at each wavelength for the three sample dilutions at both 0.5mm and 0.2mm path length.

Table 1. Absorbance values for the green food coloring taken in multiwavelength mode.

 

1 in 10 1 in 50 1 in 100

 

0.5mm 0.2mm 0.5mm 0.2mm 0.5mm 0.2mm
ID 428nm 640nm 428nm 640nm 428nm 640nm 428nm 640nm 428nm 640nm 428nm 640nm
1 0.652 0.583 0.258 0.232 0.133 0.121 0.053 0.048 0.066 0.061 0.028 0.026
2 0.651 0.589 0.261 0.235 0.132 0.121 0.054 0.048 0.066 0.061 0.027 0.025
3 0.652 0.583 0.260 0.234 0.135 0.122 0.054 0.050 0.067 0.062 0.028 0.025
4 0.650 0.582 0.263 0.239 0.133 0.121 0.054 0.049 0.067 0.063 0.028 0.025
5 0.649 0.581 0.260 0.233 0.134 0.124 0.053 0.049 0.066 0.062 0.028 0.026
6 0.650 0.592 0.262 0.236 0.133 0.121 0.054 0.050 0.066 0.063 0.028 0.026
7 0.649 0.590 0.262 0.235 0.133 0.119 0.054 0.049 0.066 0.061 0.029 0.026
8 0.649 0.587 0.261 0.234 0.133 0.124 0.054 0.048 0.066 0.062 0.028 0.026
9 0.649 0.588 0.264 0.239 0.132 0.121 0.054 0.050 0.066 0.063 0.030 0.029
10 0.651 0.591 0.261 0.234 0.133 0.121 0.054 0.051 0.066 0.061 0.029 0.027
Average 0.650 0.587 0.261 0.235 0.133 0.122 0.054 0.049 0.066 0.062 0.028 0.026
SD 0.0012 0.0040 0.0017 0.0023 0.0009 0.0015 0.0004 0.0010 0.0004 0.0009 0.0008 0.0012
Range 0.003 0.011 0.006 0.007 0.003 0.005 0.001 0.003 0.001 0.002 0.003 0.004
%CV 0.19% 0.69% 0.65% 0.99% 0.66% 1.24% 0.78% 2.10% 0.64% 1.41% 2.91% 4.59%

The results can be summarized as follows:

  • The absorbance for ten samples reads at 0.5mm path length ranged only 0.003 or less at 428nm.
  • Variation was only slightly greater at 640nm even though the absorbance peak was sharper.
  • Measurements at 0.5mm and 0.2mm path length were consistent with the path length ratio (2.5).
  • The ratio of absorbance measurements deviates slightly below 0.1A due to the increased contribution of background effects at low absorbance values; hence it is suggested that the sample has a minimum absorbance of 0.05 for accurate determination. Absorbance values for the dilutions were linear for all measurements as shown in Figure 2.

Figure 2. Linearity of absorbance measurements.

dsDNA

It is possible to determine dsDNA either using the dsDNA mode or 260/280 mode. In dsDNA mode, the DNA concentration is calculated using the formula:

Concentration (Mg/ml) = (A260-A320)*F1

where F1 = 50. Another factor to allow for the chosen path length is also automatically determined. The 320nm correction is optional but is strongly recommended while using the micro volume accessory to correct for any background interference from the sample.

DNA concentration is determined using the 260/280 mode with the following formula:

Concentration (Mg/ml) = (F1*(A1-A4))-(F2*(A2-A4))

where: A1 = 260nm, A2 = 280nm, A3 = 230nm, A4 = 320nm, F1 = 62.9 and F2 = 36.

Data for a dsDNA sample measured at 0.5mm and 0.2mm path length in dsDNA mode with 320nm correction is shown in Table 2. The sample is then diluted 1 in 50 and determined again in dsDNA mode.

Table 2. dsDNA sample concentration determination using dsDNA mode.

 

0.5mm 0.2mm 0.5mm (1 in 50)
ID A260 µg/ml A260 µg/ml A260 µg/ml
1 0.337 336.6 0.135 337.3 0.006 5.959
2 0.336 336.4 0.134 335.6 0.007 6.639
3 0.336 335.9 0.134 333.9 0.006 6.269
4 0.338 338.4 0.133 332.7 0.006 6.169
5 0.336 335.8 0.136 339.0 0.007 6.767
6 0.337 336.8 0.134 334.8 0.006 6.200
7 0.335 334.9 0.135 337.2 0.006 5.919
8 0.339 339.0 0.135 338.2 0.006 5.668
9 0.340 340.1 0.137 342.4 0.007 6.791
10 0.336 336.4 0.136 339.8 0.007 6.533
Average 0.337 337.0 0.135 337.1 0.006 6.291
SD 0.002 1.617 0.001 2.932 0.001 0.382
%CV 0.46% 0.48% 0.89% 0.87% 8.07% 6.08%
Range 0.005 5.2 0.004 9.7 0.001 1.123

The observations are as follows:

  • Absorbance range over the ten readings was 0.005 or less for each set of measurements.
  • Even at very low DNA concentration, %CV was around 6%, with an absorbance range at the very minimum of 0.001 units.
  • The value calculated for the concentration of the sample was the same at both 0.5 and 0.2mm path length showing the accuracy of the instrument.

The results for the same sample measured at 0.5mm path length using the 260/280 mode is shown in Table 3.

Table 3. dsDNA sample concentration determination using A260/A280 mode.

ID A260 A280 A230 A320 Hg/ml
1 0.337 0.198 0.148 0.001 280.7
2 0.340 0.200 0.150 0.001 282.8
3 0.337 0.199 0.148 0.001 280.9
4 0.341 0.202 0.153 0.003 282.6
5 0.344 0.203 0.152 0.001 285.7
6 0.341 0.200 0.149 0.001 283.9
7 0.340 0.201 0.149 0.002 282.0
8 0.338 0.199 0.149 0.001 280.8
9 0.344 0.203 0.152 0.003 285.3
10 0.340 0.201 0.149 0.002 282.4
Average 0.340 0.201 0.150 0.002 282.7
SD 0.002 0.002 0.002 0.001 1.792
%CV 0.73% 0.85% 1.20% 52.70% 0.63%
Range 0.006 0.004 0.003 0.002 4.95

The oligonucleotide and RNA concentration modes use the following formula:

Concentration (Mg/ml) = (A260-A320)*F1

where F1 is 30 for oligonucleotides and 40 for RNA. It is possible to determine concentration with or without correction at 320nm. Table 4 presents the results from these experiments.

Table 4. Determination of oligonucleotide and RNA sample concentration.

 

Oligonucleotide RNA

 

0.5mm 0.2mm 0.5mm 0.2mm
ID A260 µg/ml A260 µg/ml A260 µg/ml A260 µg/ml
1 0.124 74.23 0.050 75.70 0.228 182.8 0.092 183.1
2 0.124 74.66 0.051 76.59 0.228 182.7 0.092 183.7
3 0.126 75.51 0.051 76.79 0.228 182.8 0.092 183.6
4 0.126 75.42 0.050 75.20 0.227 182.0 0.093 185.6
5 0.124 74.21 0.052 77.32 0.230 184.1 0.093 185.1
6 0.123 73.93 0.051 77.23 0.229 183.0 0.092 183.6
7 0.124 74.29 0.051 76.94 0.230 183.9 0.093 185.7
8 0.123 74.07 0.053 78.95 0.230 184.2 0.093 185.8
9 0.124 74.24 0.052 77.47 0.228 182.2 0.093 185.1
10 0.124 74.63 0.053 78.89 0.228 182.5 0.092 183.8
Average 0.124 74.52 0.051 77.11 0.229 183.0 0.093 184.5
SD 0.001 0.55 0.001 1.19 0.001 0.78 0.001 1.04
%CV 0.83% 0.73% 2.09% 1.55% 0.47% 0.43% 0.57% 0.56%
Range 0.003 1.580 0.003 3.75 0.003 2.2 0.001 2.7

At both 0.5 and 0.2mm path lengths the values calculated for the concentration of the samples was the same. The absorbance range between all ten samples was 0.003 or less.

Protein

Readings at 280 and 260nm are required by the protein direct UV method and the results are calculated using the formula:

Protein concentration (Mg/ml) = (F1*(A1-A4)-(F2*(A2-A4))

where A1 = 280nm, A2 = 260nm, A3 = 230nm, A4 = 320nm, F1 = 1550 and F2 = 760.

Table 5. Absorbance readings taken using the protein direct UV mode.

ID 280 260 230 320 Hg/ml
1 0.059 0.034 0.429 0.002 1282
2 0.060 0.034 0.435 0.002 1305
3 0.060 0.035 0.439 0.002 1290
4 0.060 0.035 0.432 0.003 1274
5 0.061 0.035 0.431 0.004 1289
6 0.060 0.036 0.430 0.003 1272
7 0.059 0.035 0.428 0.003 1251
8 0.064 0.039 0.439 0.006 1281
9 0.059 0.033 0.426 0.002 1284
10 0.060 0.034 0.431 0.003 1285
Average 0.060 0.035 0.432 0.003 1281
SD 0.0015 0.0016 0.0044 0.0012 14
Range 0.005 0.006 0.013 0.004 53.5
%CV 2.45% 4.67% 1.02% 41.57% 1.09%

According to the results in Table 5 it is seen that even at low concentrations, consistent absorbance readings were obtained with an absorbance range of 0.005 at 280nm.

The formula used for calculating the protein concentration also uses the values measured at 260 and 320nm (optional) so even very small differences can have a significant effect on the calculated result.

Tips for Obtaining Reproducible Results

The following tips need to be followed to obtain repeatable readings.

  • The sample must be mixed well and free of air bubbles or particles
  • It is important to measure each sample droplet only once as the reading head moves into a default position after each sample has been measured
  • To ensure that factors which may interfere with a reading such as contaminants or sample turbidity are minimized, a reading should also be made at a second reference wavelength (where the absorbance of the sample is very low and unchanging) so as to perform a background correction.
  • In the nucleic acid and protein direct UV modes this option is defaulted to ON at a wavelength of 320nm; this can be deactivated if required.

Conclusions

The repeatability data obtained in these studies show that the Jenway Genova Nano is a highly precise instrument when using a variety of different sample types and experimental modes. Accuracy between path lengths is also apparent as is the stability of the instrument when measuring at very low sample concentrations.

About Bibby Scientific

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This information has been sourced, reviewed and adapted from materials provided by Bibby Scientific Ltd.

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Date Added: Feb 7, 2014 | Updated: Feb 7, 2014
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