Tecan NanoQuant Plate Manual de usuario

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

4 Quick Guide NanoQuant PlateTM No.30035094 Rev No. 1.4 2013-06

1.3 Computer Requirements

The following computer requirements are needed to use the i-control software:

Minimum Recommended

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

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!

2013-06 Quick Guide NanoQuant PlateTM No.30035094 Rev No. 1.4 5

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.

6 Quick Guide NanoQuant PlateTM No.30035094 Rev No. 1.4 2013-06

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.

2013-06 Quick Guide NanoQuant PlateTM No.30035094 Rev No. 1.4 7

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

8 Quick Guide NanoQuant PlateTM No.30035094 Rev No. 1.4 2013-06

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.

2013-06 Quick Guide NanoQuant PlateTM No.30035094 Rev No. 1.4 9

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.

10 Quick Guide NanoQuant PlateTM No.30035094 Rev No. 1.4 2013-06

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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