Userguide

Martin Armbrecht, Eppendorf AG, Hamburg, Germany

No 031 I BioPhotometer plus

Application of Hellma TrayCell or Implen LabelGuard microliter measurement cells in the Eppendorf BioPhotometer plus for nucleic acid determinations
Abstract
The application of microliter measurement cells from Hellma and Implen together with the BioPhotometer plus allows for the determination of very high DNA and RNA concentrations in extremely small volumes. Due to the simple handling and the convenient evaluation, this offers a low-cost alternative in comparison with determination using spectrophotometers, which are also able to perform measurements in this range. The primary purpose of this Userguide is to optimize the handling of the microliter measurement cells in order to achieve reproducible results.

Introduction
After isolation and purification of DNA and RNA, it is often necessary to determine the concentration of the nucleic acid for downstream applications. This is usually performed using a spectrophotometer. Since isolated nucleic acids are mostly highly concentrated and therefore above the measurement range of the photometer, it is first necessary to dilute the samples. The individual dilution steps have to be performed very accurately, otherwise the calculation of the starting concentration may quickly become incorrect. It is also problematic that the diluted samples mostly cannot be used in downstream applications afterwards. A convenient alternative is provided by the possibility of measuring the samples directly without any additional dilution using microliter measurement cells in combination with the BioPhotometer plus. Only a few microliters (0.7 l 5 l) are sufficient for the measurement. With this method highly concentrated samples are measured using a very short light path, e.g., 0.2 mm or 1 mm instead of the usual 10 mm light path. With a shorter light path at a certain wavelength, light can pass through the sample even at high concentrations and measurements can reproducibly be performed. Since the BioPhotometer plus does not require warm-up times, the measurement cells can directly be used for DNA or RNA measurements after switching on the device.

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Measuring principle
Figure 1 depicts an overview of the measuring principle. The light (a) of the photometer is guided into the interior of the measurement cell through the window (e), it is then directed upwards via a mirror (b) and is guided through another window (e) into the sample (c). In the lid the light is guided via a mirror (b) back to the measurement cell and transmitted via another mirror out of the cuvette. The detector of the photometer measures the remaining amount of light (absorption). The optical path length (f) that is important for the measurement is derived from the distance between the mirror and the upper exit window of the measurement cell and is important for the determination of the sample concentration. The optical path length is determined by the lid: depending on the concentration and the sample volume, lids with light path of 0.2 or 1 mm can be used. The microliter measurement cells are always delivered with both lids (Figure 2a or 2b), with labeling indicating the usage of the corresponding light path on the TrayCell lid or LabelGuard [1].

Figure 1: Schematic representation of the micro cell from Hellma (TrayCell) or Implen (LabelGuard). a) Light, b) Mirror c) Sample, d) Lid, e) Window f) Optical path length.

2a) Lid for 0.2 mm light path

2b) Lid for 1 mm light path

Concentration range
For both the 0.2 mm and the 1 mm lid it is recommended to work with an extinction range between 0.1 and 1.5 E. Table 1 shows concentration ranges for different nucleic acids in comparison to a standard cuvette with a 10 mm light path.
Table 1: Concentrations of nucleic acids by application of recommended extinction ranges (0.1 1.5 E) Nucleic acid dsDNA ssDNA RNA Oligo Factor 50 37 40 33 1 mm lid [ng/l] 50 - 750 37 - 555 40 600 33 - 495 0.2 mm lid [ng/l] 250 3750 185 2775 200 3000 165 2475 Cuvette with 10 mm light path [ng/l] 5 75 3.7 55.5 4 60 3.3 49.5

As can be seen in Table 1, when using microliter measurement cells significantly higher concentrations can be determined than in cuvettes with a 10 mm light path.

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Settings of the BioPhotometer plus

The microliter measurement cells can be directly applied, without dilution factors. The prerequisite is the adjustment of the value for the optical path length of the lid in the parameter settings at the Biophotometer plus. The settings will be saved only for the particular method applied. For the calculation of the concentration the applied light path will be automatically taken into account. Figure 3 shows the necessary adjustment of the Biophotometer plus for the direct usage of the 0,2 mm or 1mm lid.

Adjustment with the BioPhotometer 6131

When using the BioPhotometer 6131, the optical path length of 0.2 mm cannot be directly set on the device. To use the device without the need of conversion a virtual 50-fold dilution can be entered while using the preset light path of 10 mm. For this, press dilution and enter 1 l for sample and 49 l for diluent. These settings also lead to a correct result.

a)

Handling and cleaning

It is recommended to wear gloves during measurements to avoid contamination of the windows for light entry or exit. Before each measurement the cuvette should always be pressed as far as it will go into the cuvette chamber. Discrepancies in regard to the position of the measurement cell in the cuvette chamber may lead to measurement differences. Like for measurements in standard cuvettes with 10 mm light path, frozen samples should always be completely thawed and then sufficiently mixed. Between two measurements the mirror in the lid as well as the window in the upper side of the measurement cell have to be cleaned. For this purpose conventional cutton swabs or usual tissue paper may be used. Remaining debris, lint or dust should be removed using compressed air. Compressed air is available in office supply stores. In case of stronger contamination the mirror at the inner side of the lid (Figure 1a, b) and the window in the upper side (Figure 1a, e) have to be cleaned with 60% isopropanol. Further information regarding cleaning can be obtained from Hellma (www.hellma-worldwide.com).

b)

Figure 3: Adjusting the light path to 0.2 mm (a) or 1 mm (b) (e.g., for dsDNA). Depending on the lid used, the values for the corresponding optical path length have to be modified in the parameter mode.

To alter the value of the optical path length in the device settings, choose a method from a method group (e.g., the method dsDNA in the group DNA). When the arrow in the display points to the desired method, press parameter within the operating field. Press the keys with the arrows until cuvette is displayed and choose the optical path length (see Figure 3).

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Volumes
The manufacturers recommend using volumes of 3 - 5 l for the 1 mm lid and 0.7 - 3 l for the 0.2 mm lid, respectively. When using the 0.2 mm lid together with very small liquid volumes like, e.g., 1 l, the formation of small air bubbles within the light path may occur, causing variations during measurements of the same sample. For very small volumes below 2 l it is therefore recommended to pipette the sample directly onto the mirror in the inner side of the lid (Fig. 4b), instead of the usual pipetting on the upper measurement window (Fig. 4a). Since the work presented here is always performed with very small volumes, the pipetting steps should be very accurate. Of course, this is also true for pipetting the blank solutions.

Measuring Procedure
1. Wear gloves during the whole measurement procedure to avoid contamination of the measurement cell. 2. For your measurement, select a lid with the optical path length that best corresponds to the concentration range of your sample (see Table 1). 3. Choose the measurement method (e.g., dsDNA). 4. If needed, change the preset parameter in BioPhotometer plus (see setting of the BioPhotometer plus, Fig. 3a or Fig. 3b). 5. Determine the zero value (blank) with the liquid, which was used to dissolve or to dilute your sample. For this purpose pipette the blank onto the upper measurement window of the sample (Fig. 4a) or use the 0.2 mm light path on the mirror in the inner side of the lid (Fig. 4b).

Optical path length

Figure 5: Entry display before the blank measurement

a)

b)

Figure 6: Blank measurement

Figure 4: Loading of the measurement cell at 1 mm (a) or 0.2 mm light path (b)

6. Before the measurement of the blank the preset parameters like the applied light path are displayed on the screen (Fig. 5). Place the lid onto the measurement cell corresponding to the notch on the lid and press the blank key on the BioPhotometer plus (Fig. 6).

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7. The result of the blank measurement should be 0 (see Figure 7).

10. Apply your sample as shown in Fig. 4 for the blank measurement and press the Sample key (Fig. 10). The result (Fig. 11) will be displayed automatically. The applied light path will be automatically included.

Figure 7: Result of the blank measurement

8. Next, remove all liquid remnants from the inner side of the lid (Fig. 8a) using cutton swabs or tissue paper (Fig. 8b).

a)

b)

Figure 10: Pressing the Sample key

Figure 8: Cleaning of the upper measurement window (a) and the inner side of the lid (b)

9. Remove debris, lint or dust from the mirror (Fig. 9a) and the measurement window (Fig. 9b) using a compressed air sprayer, if necessary.

Figure 11: Example of a result

a)

b)

11. (Optional) If necessary the sample can be recovered with a pipette from the measurement window on top. 12. Next, remove all liquid remnants from the inner side of the lid using a cutton swabs or a tissue paper (Fig. 8b). 13. If needed, clean both areas additionally with 60% isopropanol.

Figure 9: Removing debris, lint or dust from the inner side of the lid (a) and the upper measuring window (b) with compressed air

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References
[1] C. Voolstra, A. Jungnickel, L. Borrmann, R. Kirchner, A. Huber Spectrophotometric quantification of nucleic acids - LabelGuardTM allows quantification of sample amounts in submicroliter range with commercial spectrophotometers. Application Note Implen GmbH, www.implen.com

Ordering Information Eppendorf

Product BioPhotometer plus 230 V / 50-60 Hz Thermoprinter DPU 414, incl. mains adapter and printer cable 230 V Thermo paper, 5 rolls Order no. International 6132 000.008 6131 011.006 0013 021.566 Order no. North America 952000006 952010140 952010409

Ordering Information Hellma GmbH & Co. KG

Product Hellma TrayCell, 68.5 mm, 8.5 mm, (incl. lid for layer thickness of 0.2 mm and 1 mm) Lid for TrayCell, optical path length of 1.0 mm Lid for TrayCell, optical path length of 0.2 mm Order no. International 105.800-UVS Z 8.5 mm 665.703 665.704

Ordering Information - Implen GmbH

Product LabelGuard Microliter Cell for measurement, pack with 0.2 and 1 mm path length lid, 0.7 - 5 l sample volume LabelGuard Microliter Cell for measurement, pack with 1 mm path length, 3-5 l sample volume LabelGuard Microliter Cell for measurement, pack with 0.2 mm path length, 0.7 - 4 l sample volume
Implen is a registered trademark of Implen GmbH LableguardTM is protected trademark of Implen GmbH Hellma is registered trademark of Hellma GmbH & Co. KG TrayCellTM is protected trademark of Hellma GmbH & Co. KG

Order no. International LG100UV-G LG100UV LG101UV

eppendorf is a registered trademark. All rights reserved, including graphics and images. AU03 139 020/GB1/0309/0T/KW

Your local distributor: www.eppendorf.com/worldwide Eppendorf AG 22331 Hamburg Germany Tel: +49 40 538 01-0 Fax: +49 40 538 01-556 E-mail: eppendorf@eppendorf.com Eppendorf North America, Inc. One Cantiague Road P. O. Box 1019 Westbury, N.Y. 11590-0207 USA Tel: +1 516 334 7500 Toll free phone: +1 800 645 3050 Fax: +1 516 334 7506 E-mail: info@eppendorf.com Application Support Europe, International: Tel: +49 1803 666 789 E-mail: support@eppendorf.com North America: Tel: +1 800 645 3050 ext. 2258 E-mail: support_na@eppendorf.com Asia Pacific: Tel: +60 3 8023 6869 E-mail: support_asiapacific@eppendorf.com