tecan NanoQuant Plate User manual

NanoQuant Plate™
Quick Guide

2 Quick Guide NanoQuant PlateTM No.30035094 Rev No. 1.4 2013-06
Table of Contents
1. General Information ...................................................................... 3
1.1 Introduction....................................................................... 3
1.2 Contents of the NanoQuant Plate Package.................... 3
1.3 Computer Requirements.................................................. 4
1.4 System Requirements ...................................................... 5
1.5 Applications ...................................................................... 6
2. Measurement Procedure............................................................... 7
2.1 Software Installation Procedure...................................... 7
2.2 Performing a Measurement ............................................. 7
2.3 File menu ......................................................................... 15
2.4 Quality Control of NanoQuant Plate ............................. 17
3. NanoQuant Plate.......................................................................... 19
3.1 Parameters ...................................................................... 19
3.2 Handling and Cleaning................................................... 21
3.3 Applying Samples........................................................... 22
3.4 NanoQuant Plate Disinfection ....................................... 23
4. Calculations ................................................................................. 25
4.1 Calculation of Nucleic Acid Concentration.................. 25
5. About the Quick Guide................................................................ 29

2013-06 Quick Guide NanoQuant PlateTM No.30035094 Rev No. 1.4 3
1. General Information
1.1 Introduction
Tecan’s NanoQuant Plate is intended as a general laboratory measurement
tool for the quantification of small volumes (2μl) of nucleic acids in absorbance
mode and the measurement of the labeling efficiency of nucleic acids labeled
with fluorescent dyes. The NanoQuant Plate permits the application and
parallel measurement of 16 different samples in a single measurement
procedure. After the measurement, which is controlled by Tecan’s i-control
software, the calculation of nucleic acid content and purity check using the
260/280 ratio is performed automatically and the results are displayed in an
Excel sheet. A blanking measurement, including an integrated reference
wavelength at the beginning of the measurement procedure, functions
simultaneously as a quality control check for the entire plate and indicates any
pipetting or cleaning mistakes. The plate has been designed to meet the
requirements of research laboratories working with various types of low-volume
samples including fluorophore-labeled nucleotides.
1.2 Contents of the NanoQuant Plate Package
The NanoQuant Plate package for Infinite reader series contains the following
items:
•NanoQuant Plate
•Pipetting Aid
•Safety Certificate
•This Quick Guide
•Storage Box
The NanoQuant Plate is available for the following readers:
•Infinite M200 PRO
•Infinite F200 PRO
•Infinite M200 PRO NanoQuant
•Infinite F200 PRO NanoQuant
•Infinite M1000
•Infinite F500

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1.3 Computer Requirements
The following computer requirements are needed to use the i-control software:
Minimum Recommended
PC
Windows XP/Vista (32 bit)/Windows 7 (32 or 64 bit)
Windows compatible PC with a Pentium compatible
processor running at 1 GHz
2 GHz
(Dual Core)
Operating
System
Windows XP (32-bit) SP3
Windows Vista (32-bit)
Windows 7 (32-bit)
Windows 7 (64-bit)
Windows XP
(32-bit) SP3
Memory
Windows XP: 512 MB RAM
Windows Vista (32-bit): 1 GB RAM
Windows 7 (32-bit): 1 GB RAM
Windows 7 (64-bit): 2 GB RAM
1 GB RAM
2 GB RAM
2 GB RAM
3 GB RAM
Space
Requirements 700 MB 1 GB
Monitor Super VGA Graphics
Resolution 1024 x 768 1280 x 1024
Color Depth 256
Mouse Microsoft mouse or compatible pointing device
Communication 1 x USB 2.0
2 x USB 2.0,
1 x RS232
(Serial)
Devices
1 x CD-ROM drive
Windows Vista: DirectX 9 graphics and 32 MB of
graphics memory (for Home Basic); 128 MB of
graphics memory plus WDDM support for all other
versions
Windows 7: DirectX 9 graphics device with WDDM 1.0
or higher driver
.NET
Microsoft .NET Framework 2.0
If this version is not present, the install/upgrade
program will install it side-by-side with any existing
installations of the .NET Framework.
Windows
Installer
3.1
If this version is not present, the install/upgrade
program will install it.
Microsoft Excel 2002, 2003, 2007, 2010 (32-bit) – Starter edition NOT
supported!

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1.4 System Requirements
To perform a NanoQuant measurement, the following items are required:
Infinite 200 Pro
•An Infinite 200 Pro reader with firmware version V2.0 or higher
•A computer with i-control V1.4 Service Pack 1 or higher installed
•The NanoQuant Plate Package
Make sure the following absorbance filters are available on the filter slide of
your Infinite F200 PRO:
•Position 1: 260 nm (5 nm bandwidth)
•Position 2: 280 nm (3 nm bandwidth)
•Position 3: 340 nm (10 nm bandwidth)
•Position 4: free (for individual use)
For Infinite F200 PRO, the filter positions must remain in the order in which they
were delivered. The original filter positions guarantee the fastest filter switching
for well-wise measurements. The Infinite M200 PRO can be used immediately
for measurement without any calibration of the monochromator.
Infinite M1000, Infinite F500
•An Infinite M1000 reader (REF 30061442) with main firmware version
V2.0 or higher
•An Infinite F500
•Software: i-control V1.8 or higher
•The NanoQuant Plate Package
Make sure the following absorbance filters are available on the filter slide of
your Infinite F500:
•Position 1: 260 nm (5 nm bandwidth)
•Position 2: 280 nm (3 nm bandwidth)
•Position 3: 340 nm (10 nm bandwidth)
Note
Only use the Infinite reader and the NanoQuant Plate at room temperature and
under normal laboratory conditions.

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1.5 Applications
Nucleic Acid Quantification (Infinite 200 PRO, Infinite M1000,
Infinite F500)
For the quantification procedure in the NanoQuant Plate, a sample volume of
2 μl is sufficient for accurate results. Absorbance of nucleic acid samples is
measured at 260 nm. The optical path length of the NanoQuant Plate is
0.5 mm. To assess the purity of the nucleic acid, an additional measurement at
280 nm is performed to indicate proteins in the sample. For pure nucleic acids,
a 260/280 ratio between 1.8 - 1.9 is acceptable. If this ratio is lower than 1.8 it
may indicate the presence of proteins or other contaminants. If this is the case,
an additional purification step/procedure is recommended.
Labeling Efficiency (Infinite M200 PRO and Infinite M1000 only)
Working with nucleic acids labeled with fluorescent dyes requires samples of
high quality. Besides common nucleic acid quantification and nucleic acid purity
check with 260/280, the labeling efficiency is an important criterion for improved
research results. With the NanoQuant Plate it is possible to measure
absorbance of nucleic acids labeled with Cy3, Cy5, Alexa 555, Alexa 647 and
many other fluorescent dyes.
Note
All measurements on Infinite M1000 involving NanoQuant Applications are
performed using one measurement channel only.

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2. Measurement Procedure
2.1 Software Installation Procedure
The i-control software is installed using the following procedure:
1. Insert the i-control software CD ROM into your CD ROM drive.
2. A window opens with different selectable options.
3. Choose Software and install i-control.
4. Follow the instructions of the Wise Installation Wizard.
5. When installation is successful, exit the Installation window.
6. Switch on and connect to Infinite instrument.
Note
i-control is delivered with the Infinite reader series.
2.2 Performing a Measurement
For applications using the NanoQuant Plate, a tab called Applications is
implemented in i-control software, so that all measurements can be
performed quickly and easily.
1. Start i-control.
2. Connect to the Infinite instrument. The standard i-control window
opens.
3. Select Applications in the lower left part of the window.
Figure 1: Overview of i-control script

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4. Select the desired measurement type in the control bar on the left side
of the window by double-clicking or dragging and dropping:
•Nucleic acid quantification (Infinite 200 PRO, Infinite M1000,
Infinite F500)
•Labeling Efficiency (Infinite M200 PRO and Infinite M1000 only)
5. The corresponding measurement stripe appears and the NanoQuant
Plate definition file (NanoQuant Plate Tecan 16 Flat Black) is
automatically selected in the Plate field.
6. Select blanking mode. Select the Individual Blanking check box for
individual blanking or leave the check box clear for average blanking.
See Individual Blanking and Average Blanking on page 12.
7. Depending on the connected instrument, the wavelengths used for
measurement are selected automatically (make sure that the correct
filters are properly installed and defined on the filter slides of the
Infinite F200 PRO and Infinite F500 instrument).
Instrument Bandwidth at 260 nm Bandwidth at 280 nm
Infinite M200 PRO: 5 nm 5 nm
Infinite F200 PRO: 5 nm 3 nm
Infinite M1000: 5 nm 5 nm
Infinite F500: 5 nm 3 nm
8. Select a sample type (e.g. dsDNA, ssDNA, RNA, etc. in the Sample
type drop-down list.
9. In addition, select the respective dye(s) in the Markers drop-down lists
for Labeling Efficiency measurements. If the samples are labeled with
only one fluorophore, set the drop-down list of Dye 2 to None.
10. When all settings are correct, click the Start Blanking button to
initialize the blanking measurement. The plate transport moves out
and the user is requested to insert the NanoQuant Plate with the
respective blanking buffer.

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Figure 2: Correct orientation of NanoQuant Plate in the reader
11. The first measurement step is blanking with the required buffer. A
separate window opens and the blanking procedure can be observed.
12. The blanking measurement is started and can be monitored in the
measurement progress window. If blanking has been performed
successfully, the sample positions are highlighted in yellow and the
screen color changes to a homogenous green (Nucleic Acid
Quantification) or blue (Labeling Efficiency). Blanking results (date and
time, samples positions that were selected for blanking, blanking
range, and maximum CV) are displayed next to the plate preview in
the measurement stripe and saved until the instrument is
disconnected.
13. When the blanking measurement has been completed successfully,
the plate is moved out automatically. The plate is now ready for
sample application and analysis. The green Start button is now
accessible.
14. Remove any remaining blanking buffer from the sample positions by
wiping the quartz spots with a piece of lint free paper and apply 2 μl of
the samples onto each spot.
15. When the NQP is loaded with samples and correctly placed onto the
plate carrier, click the green Start button.

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16. As the measurement is performed an Excel sheet opens automatically
in the background. All measurement results (including the
automatically calculated nucleic acid concentration, the 260/280 ratio,
and for Labeling Efficiency measurements, the dye concentration)are
concisely displayed in a matrix (analogous to the plate layout). The
OD values of each sample at all relevant wavelengths are also
displayed.
Figure 3: Overview of Excel result sheet
17. Once the measurement procedure is finished, the plate is moved out
automatically. A pop-up message appears, asking if the user wishes
to perform another measurement.
•If additional (identical) measurements are to be performed, wipe
oany sample residues from the previous measurement and
apply new samples. Click Yes to start the measurement.
•If no further measurement is to be performed, click No. An extra
sheet appears in the Excel workbook summarizing the results of
all previous measurements.

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Figure 4: Automatic summary in Excel of all measured samples
18. When all measurements are completed, clean the NanoQuant Plate
thoroughly and store it appropriately.
19. Once i-control has been closed, the Infinite reader can be switched
off.
Note
Excel workbooks containing the measurement results are not saved
automatically. This has to be done by the user. It is possible to save the
method script created in i-control for further use by clicking Save in the File
menu.

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Additional blanking information
The blanking information will be stored until:
•The reader is disconnected
•The i-control is shut down
•Another sample type is selected
If other sample types are selected, the screen color will become a light
green or light blue gradient again depending on the application and the
blanking procedure must be repeated with corresponding wavelengths.
If you have chosen a labeling efficiency measurement before starting a
blanking procedure, the type of samples and the specific wavelengths for
dyes have to be selected. If the correct fluorophores are selected, the
samples can be applied into the NanoQuant Plate.
Individual Blanking and Average Blanking
The user may choose between two distinct blanking options: Average
Blanking and Individual Blanking (set by default). For Average Blanking,
choose which wells are to be used for blanking by dragging a frame around
the respective sample positions in the plate preview. It is generally
recommended to perform the blanking measurement with all 16 sample
positions, however average blanking must be done with at least two wells,
in order to calculate an average value that is used to blank-correct all
measured samples.
The OD results measured with average blanking have to be below 10% CV
in order to grant reliable measurement results.
Figure 5: Click Start Blanking button for Average Blanking

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In contrast, Individual Blanking requires blanking on all wells that are to be
used for subsequent measurements. Individual blanking information is
stored for each well used and blank-correction of the samples is done with
the corresponding single blanking value (of the same well) instead of one
average blank.
Figure 6: Select Individual Blanking and click the Start Blanking button to start
individual blanking
It is recommended to use Individual Blanking as the standard blanking
option in order to obtain the most precise and reliable results.
Figure 7: Individual Blanking information in the Excel result sheet
Note
The CV value displayed in the Last Blanking box (see Figure 11) represents
the variation coefficient of all wells used for blanking.

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Sample ID function
The Sample ID function allows the user to individually name each
sample/well. By clicking the Sample ID box, a window opens and the user
may enter the desired sample names. By clicking the Clear button, all
inserted Sample IDs are deleted. Sample IDs are displayed in the summary
sheet. Click Save to store the entries and click Close to complete the
procedure.
Sample IDs may also be copied from Excel sheets and pasted into sample
ID list.
“Show Raw Data”
To view all measurement results as raw values, the NanoQuant tab in the
Results Presentation dialog box contains the Show Raw Data check box.

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By checking this box, the results summary sheet will include raw OD values
at all measured wavelengths as well as blank- and reference wavelength-
corrected OD values and the automatically calculated sample
concentrations and ratio values.
Figure 8: Raw Data Output in the Excel results summary
(1) Nucleic acid quantification
(2) Labeling efficiency
2.3 File menu
i-control Toolbar
Start a measurement
Connect or disconnect an instrument
Move plate out
Move plate in
Show or hide the info pane
Open the i-control help file.
Sample
The following sample types can be selected:
•dsDNA
•ssDNA
•RNA
•Others

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Upon selecting Others, the Edit Samples window opens. The star marks a
free line where additional sample types can be entered with the
corresponding standard coefficient [µg/ml] for 1 cm path length at 260 nm.
A ratio wavelength of 230 nm or 280 nm can also be selected from the
drop-down menu.
Figure 9: Selecting and adding new samples
Note
For the Infinite F200 PRO and Infinite F500, the correct filters must always
be used and defined correctly in the filter slide. If a wavelength is selected
that is not available (because the filter is not in the slide) an error message
appears.
Figure 10: Editing or creating new markers
Note
The selected sample in Edit Sample must always have the ratio wavelength
set at 280 nm, otherwise no marker can be selected for Dye 1 or Dye 2 and
an error message appears.

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2.4 Quality Control of NanoQuant Plate
Average Blanking Out of Range (CV ≥ 10 %)
If Average Blanking is out of range, the failed wells are highlighted in pink
and an error message appears requesting the user to repeat blanking
measurement.
In this case, single wells are displayed in pink, indicating these wells as
diverging after blanking. These wells differ from the calculated CV by 10 %,
meaning that these quartz wells are dirty due to lint, fingerprints, etc.
Figure 11: Color code for blanking: pink wells are out of range

18 Quick Guide NanoQuant PlateTM No.30035094 Rev No. 1.4 2013-06
There are two possibilities to complete the blanking procedure:
1. Repeat blanking with the same plate and select white wells
(e.g. E1 – G2 in the example below) by dragging a frame around
them. The newly selected wells will appear white, the diverging wells
change from pink to purple and all other wells turn blue to indicate that
they are unused.
Figure 12: Color code for blanking procedure when re-selected: purple wells indicate
formerly pink wells
2. Move the plate out, repeat the cleaning procedure and apply fresh
blanking samples into the plate and start blanking again.

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3. NanoQuant Plate
3.1 Parameters
Physical
Optics 16 quartz lenses (one per sample)
Quartz lens Optical path length: 0.5 mm diameter: 2.2 mm
Parallel measurement 16 sample positions (2 rows of 8)
Sample volume Min. 2 μl
Dimensions plate Plate width: 85.4 mm Plate height: 14.6 mm Plate length:
127.8 mm Weight: ~ 160 g
Wavelength settings
Infinite M200 PRO 260 nm (5 nm bandwidth), 280 nm (5 nm bandwidth),
310 nm (5 nm bandwidth) reference
Infinite M1000 260 nm (5 nm bandwidth), 280 nm (5 nm bandwidth),
310 nm (5 nm bandwidth) reference
Infinite F200 PRO, Infinite F500 260 nm (5 nm bandwidth), 280 nm (3 nm bandwidth),
340 nm (10 nm bandwidth) reference
Measurement time of whole plate
Quantify Nucleic Acid Infinite 200 PRO: 1.15 minute for 16 samples
Labeling Efficiency Infinite 200 PRO: 2 minutes for 16 samples

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Typical performance values Infinite 200 PRO
Wavelength absorbance
Wavelength accuracy < ± 0.5 nm for λ > 315 nm
< ± 0.3 nm for λ≤ 315 nm
Wavelength reproducibility < ± 0.5 nm for λ > 315 nm
< ± 0.3 nm for λ ≤ 315 nm
Absorbance bandwidth Infinite M200 PRO < 5 nm for λ ≤ 315 nm
< 9 nm for λ > 315 nm
Absorbance bandwidth Infinite F200 PRO Depending on filters used
Measurement absorbance
Detection limit (DNA concentration) 1 ng/μl dsDNA
Reproducibility of one sample (50 μg/ml) < 1 % CV
Ratio 260/280 (50 μg/ml) ± 0.07
Precision @ 260 nm < 0.2 %
Accuracy @ 260 nm < 0.5 %
Figure 13: Technical drawing of the NanoQuant Plate (dimensions in mm)