User Guide spectro

7/31/2019 User Guide spectro

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Application of Hellma TrayCell or Implen Label-

Guard microliter measurement cells in the Eppendorf

BioPhotometer plus for nucleic acid determinations

Martin Armbrecht, Eppendorf AG, Hamburg, Germany

No 031 I BioPhotometer plus

Userguide

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.

Abstract

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 ac-

curately, otherwise the calculation of the starting concen-

tration may quickly become incorrect. It is also problematic

that the diluted samples mostly cannot be used in down-

stream applications afterwards.

Introduction

A convenient alternative is provided by the possibility of

measuring the samples directly without any additional dilu-

tion 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 concentra-

tions 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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Userguide No 031 | Page 2

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.

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.

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.

Concentration range

Table 1: Concentrations of nucleic acids by application of recommended extinction ranges (0.1 1.5 E)

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 determina-

tion of the sample concentration. The optical path lengthis 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].

2a) Lid for 0.2 mm light path 2b) Lid for 1 mm light path

Nucleic acid Factor 1 mm lid [ng/l] 0.2 mm lid [ng/l] Cuvette with 10 mm lightpath [ng/l]

dsDNA 50 50 - 750 250 3750 5 75

ssDNA 37 37 - 555 185 2775 3.7 55.5

RNA 40 40 600 200 3000 4 60

Oligo 33 33 - 495 165 2475 3.3 49.5


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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 Biopho-

tometer plus for the direct usage of the 0,2 mm or 1mm lid.

a)

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 param-

eter within the operating field. Press the keys with the

arrows until cuvette is displayed and choose the optical

path length (see Figure 3).

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.

Settings of the BioPhotometer plus Adjustment with the BioPhotometer 6131

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 measure-

ment 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. Com-pressed 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).

Handling and cleaning


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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 BioPhoto-

meter 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).


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, respec-

tively. 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

Volumes

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

path (b)

Optical

path

length

Figure 5: Entry display before the blank measurement

Figure 6: Blank measurement

a) b)

Measuring Procedure

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).

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.


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7. The result of the blank measurement should be 0

(see Figure 7).

8. Next, remove all liquid remnants from the inner side of

the lid (Fig. 8a) using cutton swabs or tissue paper

(Fig. 8b).

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 7: Result of the blank measurement

Figure 10: Pressing the Sample key

Figure 11: Example of a result

Figure 8: Cleaning of the upper measurement window (a) and the

inner side of the lid (b)

a) b)

Figure 9: Removing debris, lint or dust from the inner side of the lid (a)

and the upper measuring window (b) with compressed air

a) b)

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.

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.


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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

eppendorfi

saregisteredtrademark.

Allrightsreserved,includinggraphicsandimages.

A

U03139020/GB1/0309/0T/KW

Ordering Information Eppendorf

Product Order no. International Order no. North America

BioPhotometer plus 230 V / 50-60 Hz 6132 000.008 952000006

Thermoprinter DPU 414, incl. mains adapter and printer cable 230 V 6131 011.006 952010140

Thermo paper, 5 rolls 0013 021.566 952010409

Ordering Information Hellma GmbH & Co. KG

Product Order no. International

Hellma TrayCell, 68.5 mm, 8.5 mm, (incl. lid for layer thickness

of 0.2 mm and 1 mm)

105.800-UVS Z 8.5 mm

Lid for TrayCell, optical path length of 1.0 mm 665.703

Lid for TrayCell, optical path length of 0.2 mm 665.704

Ordering Information - Implen GmbH

Product Order no. International

LabelGuard Microliter Cell for measurement, pack with 0.2 and 1 mm

path length lid, 0.7 - 5 l sample volume

LG100UV-G

LabelGuard Microliter Cell for measurement, pack with 1 mm path length,

3-5 l sample volume

LG100UV

LabelGuard Microliter Cell for measurement, pack with 0.2 mm path

length, 0.7 - 4 l sample volume

LG101UV

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

Your local d istr ibutor: www.eppendorf .com/worldwide

Eppendorf AG 22331 Hamburg Germany Tel: +49 40 538 01-0 Fax: +49 40 538 01-556 E-mail: [email protected]

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: [email protected]

Application Support Europe, International: Tel: +49 1803 666 789 E-mail: [email protected]

North America: Tel: +1 800 645 3050 ext. 2258 E-mail: [email protected]

Asia Pacific: Tel: +60 3 8023 6869 E-mail: support_asiapacif [email protected]

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User Guide spectro