biochrom NanoVue Plus User Manual

NanoVue Plus

™

Product User Manual

Biochrom US Telephone: [phone]
84 October Hill Rd Toll Free: 1-800-272-2775
Holliston, MA Fax: [phone]
01746-1388 [e-mail]
USA www.biochromspectros.com

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5061-049 REV 1.4

1. INSTALLATION 3

1.1. Unpacking and positioning 3

1.2. Safety 3
4

2. INTRODUCTION 5

2.1. Your NanoVue Plus 5

2.2. File system 6

2.3. Data export 6

2.4. Sample treatment 7

2.5. Pathlength and Absorbance nomalization 7

2.6. Auto-Read 8

2.7. Quality Assurance 8

2.8. Keypad and display 9

2.9. Software style 10

3. OPERATION AND MAINTENANCE 11

3.1. Sample application guide 11

3.2. Sample Plate replacement 12

3.3. Cleaning and general maintenance 13

3.4. Return for repair 13

3.5. Lamp replacement 13

3.6. Replacing the printer paper 13

4. LIFE SCIENCE 14

4.1. File system 14

4.2. Nucleic acids 14

4.2.1. Theory 14

4.2.2. DNA measurement 16

4.2.3. RNA measurement 17

4.2.4. Oligonucleotide measurement 19

4.2.5. T

m

Calculation 20

4.2.6. CyDye™ measurement 23

4.3. Proteins 26

4.3.1. Theory 26

4.3.2. Protein UV 28

4.3.3. Protein A280 30

4.3.4. BCA 31

4.3.5. Bradford 34

4.3.6. Lowry 36

4.3.7. Biuret 39

5. APPLICATIONS 42

5.1. File system 42

5.2. Single Wavelength 42

5.3. Concentration 44

5.4. Wavescan 46

5.5. Kinetics 48

5.6. Standard Curve 50

5.7. Multiple Wavelength 53

5.8. Absorbance Ratio 54

6. FAVOURITES AND METHODS 56

7. UTILITIES 57

7.1. Date and time 57

7.2. Regional 57

7.3. Printer 58

7.4. Preferences 58

7.5. Contrast 58

7.6. About 58

7.7. Pathlength Check & Calibration 59

7.8. Games 62

8. ACCESSORIES 63

8.1. Printer installation 63

8.2. Bluetooth accessory installation 65

8.3. Fitting SD memory card accessory 67

8.4. After sales support 70

9. TROUBLESHOOTING 71

9.1. Pathlength calibration over-range 71

9.2. Error messages 72

9.3. Fault analysis 74

9.4. Frequently asked questions 74

10. SPECIFICATION AND WARRANTY 77

11. LEGAL

Page finder

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5061-049 REV 1.4

1. INSTALLATION

1.1. Unpacking and positioning

• Removetheinstrumentfromitspackagingandinspectitforsignsofdamage.If

any are discovered, inform your supplier immediately.

• Theinstrumentmustbeplacedonastable,levelsurfacethatcantakeitsweight
(~ 4.5 kg) and positioned such that air can circulate freely around the casing.

• Ensureyourproposedinstallationsiteconformstotheenvironmentalconditions

for safe operation.

• Theinstrumentisdesignedforindooruseonly,temperaturerange5°Cto35°C

and should be kept away from strong draughts.

• Ifyouusetheinstrumentinaroomsubjectedtoextremesoftemperature

change during the day, it may be necessary to recalibrate (by switching off and
then on again) once thermal equilibrium has been established (2–3 hours).

• Atemperaturechangeofnomorethan4°C/hourandamaximumrelative
humidityof80%at31°C,decreasinglinearlyto50%at40°Carerequired.

• Iftheinstrumenthasjustbeenunpackedorhasbeenstoredinacold

environment, it should be allowed to come to thermal equilibrium for 2–3 hours
in the laboratory before switching on. This will prevent calibration failure as a
result of internal condensation.

• Theinstrumentmustbeconnectedtothepowersupplywiththepoweradaptor

supplied. The adaptor can be used on 90–240V, 50–60Hz supplies. It will become
warm once plugged into the power supply and should not be covered up.

• Switchontheinstrumentviathekeypad(

) after it has been plugged in. The

instrument will perform a series of self-diagnostic checks.

• Itisrecommendedthatusersreadthroughthismanualpriortouse.

• Contactyoursupplierifyouexperienceanydifficultieswiththisinstrument

• Werecommendthatyoucarryoutaquickcheckofthepathlengthbeforeusing

1.2. Safety

Spectrophotometer Health & Safety Document including General Operating
Instructions are available as a booklet provided with each instrument. The booklet,
translated into the European Union languages, is available on the delivered CD. The
instructions provide the user with basic use, troubleshooting and how to use the
instrument in a safe manner.

CAUTION

This instrument contains a UV source which generates a light beam that

is present at the sample platform. Do not attempt to divert the beam as
prolonged exposure to the beam may cause permanent eye damage.

Note that the instrument prevents operation if the head is raised or the Xenon
flash does no reach the detection system.

the NanoVue, please see instructions on how to do this in section 7.7.

1. INSTALLATION

1.1. Unpacking and positioning

• Removetheinstrumentfromitspackagingandinspectitforsignsofdamage.If

any are discovered, inform your supplier immediately.

• Theinstrumentmustbeplacedonastable,levelsurfacethatcantakeitsweight
(~ 4.5 kg) and positioned such that air can circulate freely around the casing.

• Ensureyourproposedinstallationsiteconformstotheenvironmentalconditions

for safe operation.

• Theinstrumentisdesignedforindooruseonly,temperaturerange5°Cto35°C

and should be kept away from strong draughts.

• Ifyouusetheinstrumentinaroomsubjectedtoextremesoftemperature

change during the day, it may be necessary to recalibrate (by switching off and
then on again) once thermal equilibrium has been established (2–3 hours).

• Atemperaturechangeofnomorethan4°C/hourandamaximumrelative
humidityof80%at31°C,decreasinglinearlyto50%at40°Carerequired.

• Iftheinstrumenthasjustbeenunpackedorhasbeenstoredinacold

environment, it should be allowed to come to thermal equilibrium for 2–3 hours
in the laboratory before switching on. This will prevent calibration failure as a
result of internal condensation.

• Theinstrumentmustbeconnectedtothepowersupplywiththepoweradaptor

supplied. The adaptor can be used on 90–240V, 50–60Hz supplies. It will become
warm once plugged into the power supply and should not be covered up.

• Switchontheinstrumentviathekeypad(

) after it has been plugged in. The

instrument will perform a series of self-diagnostic checks.

• Itisrecommendedthatusersreadthroughthismanualpriortouse.

• Contactyoursupplierifyouexperienceanydifficultieswiththisinstrument

• Werecommendthatyoucarryoutaquickcheckofthepathlengthbeforeusing

WARNING

High voltages exist inside the NanoVue Plus instruments. Repair and
maintenance should only be carried out by individuals trained specifically to
work on these instruments.

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5061-049 REV 1.4

If the instrument is used in a manner not specified or in environmental conditions
not appropriate for safe operation, the protection provided may be impaired and
instrument warranty withdrawn.

There are no user-serviceable parts inside this instrument.

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5061-049 REV 1.4

2. INTRODUCTION

2.1. Your NanoVue Plus

NanoVue Plus is a simple-to-use UV/Visible instrument with twin CCD array
detectors (1024 pixels) and few moving parts, which contributes to its inherent
reliability.

General Principles

In conventional UV/Visible spectrophotometers, the sample is usually contained
within a glass or silica cuvette which is placed in the sample beam.
The Absorbance of this is measured and then compared to that of a standard.
From this comparison, the concentration is calculated.

However, when the quantity of sample is limited or highly concentrated, dilution ­or the use of ultra low volume cuvettes is required, which is time consuming, can
introduce errors and presents cleaning difficulties.

NanoVue Plus was developed to overcome these problems. Using NanoVue Plus,
typical sample volumes of 2 µl are pipetted onto a hydrophobic surface and then
a very short pathlength of either 0.2 mm or 0.5 mm is created by lowering the
sampling head onto the top of the sample.

With the sample so constrained, all of the general software features of the
instrument, including wavelength scanning, single or multi-wavelength
Absorbance and concentration measurement, kinetics, standard curves and
Absorbance ratio, can be utilized. In all of these modes, the instrument utilizes
either the 0.2 mm or 0.5 mm pathlengths. The benefit of these small pathlengths is
that the instrument can measure smaller volumes of very concentrated or highly
absorbing samples.

NanoVue Plus also has a range of more specific life science applications, including
DNA and RNA concentration and purity, Oligonucleotide concentration, Tm
Calculation, Cy Dye concentration and a variety of protein measurements. In all of
these applications, instead of the actual Absorbance measurement being used, the
value is normalized to reflect a standard pathlength of 10 mm, so that generally
accepted factors (50 for DNA, 40 for RNA and 33 for oligos, for example) can be
used to calculate concentration. As an example, if the absolute Absorbance of
the sample is 0.025 A using a pathlength of 0.5 mm, the normalized Absorbance
shown will be 0.500 A.

NanoVue Plus is therefore ideally suited to the life scientist where sample is limited
and speed and convenience of analysis is key.

Figure 1: NanoVue Plus
Spectrophotometer

UV-Visible

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5061-049 REV 1.4

2.2. File system

After switch on and automatic checking the instrument defaults to a home page
entitled ‘NanoVue Plus‘. This page displays five folders which form the topmost
layer of a simple file tree which is the basis of the user-interface. The folder
screens are reached by pressing the appropriate number on the keypad, with
return to the top level by means of the ‘Esc’ key. The folders group various facilities
together as follows:-

1. Life Science Standard life science applications such as nucleic acid and
protein assays.

2. Applications General spectroscopic applications.

3. A folder to store your more frequently used methods.

(Inactive when empty)

4. Methods Contains 9 folders that can store less frequently used methods.
Up to 9 methods per folder are allowed, totalling 81 methods.

5. Utilities Instrument set up options and games.

Figure 2: NanoVue Plus with Printer

Figure 2: NanoVue Plus with Printer

Favorites

Data can be transferred to a PC, either via a Bluetooth accessory (supplied pre­installed or as an optional accessory) or via a USB cable. The software to perform
this task “PVC” (Print Via Computer) is a small application running under Windows
2000™, Windows XP™ or Windows 7, 8, 8.1. PVC can operate via USB and Bluetooth
simultaneously.

PVC can store data either in a common directory or can be configured to save
to independent directories by both file format and connection. The data may be
stored as an Excel spreadsheet, an EMF graphics file, a comma delimited (csv) data
file, a tab delimited (txt) data file, rich text format (RTF) which is compatible with
Word or in native PVC format.

Some users may find it convenient to “print through” the PC directly to a printer
already attached to it. The software to enable this option will be found on the CD
provided.

Datrys Computer control software is also available for NanoVue Plus as an optional
extra.

2.3. Data export

An integral printer is available for the instrument; this may be either supplied
pre-installed or as an optional accessory. The installation procedure is described in
section 8.1.

(Games only available if selected via the Preferences Screen)

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2.4. Sample treatment

NanoVue Plus uses a unique sampling head which enables users to accurately
measure the optical characteristics of samples volumes as low as 0.5 µl. The
sample is applied to a horizontal plate having a hydrophobic surface, the
sampling head is lowered into position and the reading taken. The pathlength is

automaticallyadjustedunlesstheuserselectsmanualadjustment.Thiscanbe

either 0.2 mm or 0.5 mm.

Application of the sample is most conveniently carried out using any commercially
available pipetting tool and standard tips, a 10 µl pipette is recommended.

2.5. Pathlength and Absorbance
normalization

For methods in the Application folder, the Absorbance values have not been
normalized and the pathlength needs to be taken into account when the values
are used for concentration calculations. NanoVue Plus has the ability to measure
samples using either a 0.2 mm or a 0.5 mm pathlength. The 0.5 mm pathlength
should be used for samples of low concentration and the 0.2 mm pathlength for
more concentrated samples. It is best to use the 0.5 mm pathlength whenever
possible. In the Life Science applications, the Absorbance readings are presented
as their normalized 10 mm pathlength values, to allow the use of literature based
factors for concentration measurements.

Within the DNA, RNA, Oligonucleotide, Tm Calculation, Cy Dye, Protein UV and
Protein A280 methods the system defaults to having the pathlength chosen
automatically. When this is active, the instrument will measure at the 0.5 mm
pathlength first and if the Absorbance is high (> 1.7 A) then the measurement will
be carried out at the 0.2 mm pathlength. If the concentration range of the samples
is known beforehand, the relevant pathlength can be pre-selected; if it is unknown
then the automatic option can be used. Within the other applications, the actual
measured Absorbance value is reported, so if the Absorbance is high at the

0.5 mm pathlength (> 1.7 A), the sample can be re-measured at the 0.2 mm
pathlength. In the automatic mode, the reference scan is always carried out
using both pathlengths, so that if a sample is measured at one pathlength and
subsequently re-measured at the other pathlength, a new reference scan is not
required.

The pathlength that is used to carry out the measurement is printed/exported with
each result and is also displayed in the top left hand corner of the header line on
the instrument’s display. Care must be taken that the correct pathlength is being
used when comparing sample results or using a stored calibration curve.

Figure 3a: Sample head

Figure 3b: Sample application

3a

3b

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5061-049 REV 1.4

2.6. Auto-Read

NanoVue Plus senses when the sampling head has been lowered, so Auto-Read
can be used to automatically make a measurement when this happens, obviating
the need to press any keys. The instrument assumes that the first reading after
starting any application will be a reference scan and that subsequent readings will
be sample scans.

Switching Auto-Read on or off:

Go to Utilities (option 5 from the NanoVue Plus menu).
Select Preferences (option 4).
Use the arrow keys to go to the Lid Switch option and select On to turn Auto-Read

on, or Off to turn Auto-Read off. Press the confirm button.
When Auto-Read is off, use the 0A/100%T and

keys to take the reference and

measurement scans respectively.
When Auto-Read is on, the keys on the keypad are still functional. This enables

a user to carry out a new reference scan at any time by pressing the 0A/100%T
key prior to lowering the sampling head, or to perform a series of readings or
reference scans without raising the sampling head.

2.7. Quality Assurance

Quality Assurance is provided to help minimize the impact of pipetting errors. If
Quality Assurance is switched on from the Preferences Screen, the instrument will
ask the user for a second reference to compare with the first to ensure that the
reading is reasonable. If the second reading is not sufficiently close to the first
reading, the user will be asked to replace the reference again. The process will be
repeated until two successive good readings (within 0.02 A) are identified and the
reference will be the average of these readings.

Note: When the user is prompted for the second reference, the user is required
to clean the surface and reapply the sample. The 0A/100%T key is disabled until

theuserliftsthesamplingheadtopreventtheuserfromjustre-readingthesame

sample.

0.5 mm pathlength

0.2 mm pathlength

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5061-049 REV 1.4

Key Action

On/Off key Turns the instrument on/off.

Arrow keys Use the four arrow keys to navigate around

the display and select the required setting
from the active (highlighted) option.

View options



View options for that application mode.
Some of these are common to all applications
and are described below. Options unique to
an application are described in the relevant
section.

Alphanumeric keys Use these to enter parameters and to write

text descriptions. Use repeated key presses to
cycle through lower case, number and upper
case. Leave for 1 second before entering the
next character. Use the C key to backspace
and the 1 key to enter a space.

Escape/Cancel

Esc

Escape from a selection and return to the
previous folder.

Set Reference

OA

100%T

Set reference to 0.000 A or 100%T on a
reference solution at the current wavelength
in the mode selected. When in scan mode,
make a reference scan.

Confirm selection

Confirms a selection.

Take measurement

Makes a measurement.

2.8. Keypad and display

The back-lit liquid crystal display is very easy to navigate around using the
alphanumeric entry and arrow keys on the hard-wearing, spill-proof membrane
keypad.

OA

100%T

Esc

Esc

1

2

3

abc

def

5

6

4

mno

jkl

ghi

7

8

9

wxyz

tuv

pqrs

C

0

+ / -

A

C

G

T/U

0A

100% T

Alphanumeric

Confirm selection

Escape/Cancel

On/Off

Set reference

Take measurement

View options

Arrow keys

Display
screen

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5061-049 REV 1.4

Options Sub Menu (select using keypad numbers)

1. View Parameters for the experiment.

2. Print the results.
3, 4, 5, 6 Described in the relevant application.

7. Define the Sample Number you wish to start from.

8. Save the parameters as a method to a defined folder name with

a defined method name.

9. Toggle Auto-Print on/off. Default is off.

Exit options by pressing Esc

Esc

or wait.

Experienced operators can use the numeric keys as a shortcut to the option
required without needing to enter the Options menu.

2.9. Software style

The user interface is built around folders of files which are displayed on the first
page when the instrument is switched on. Different folders are numbered and
opened by using the associated number key on the keypad.

1. Standard life science applications such as
nucleic acid and protein assays.

2. General spectroscopic applications.

3. A folder to store your more frequently used

methods. (Inactive when empty)

4. Contains 9 folders that can store less
frequently used methods. Up to 9 methods
per folder are allowed, totaling 81 methods.

5. Instrument set up options and games.
(Games only available if selected via the

Preferences Screen)

Summary
Function Keypad Number Description

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3. OPERATION AND
MAINTENANCE

3.1. Sample application guide

Step 1

Lift the sampling head to the vertical position and using a low-volume (0–10 µl)
pipette take up approximately 2 µl of sample. A reliable pipette and matching high
quality tips are strongly recommended.

When removing a very small aliquot from a larger volume, ensure that the sample
is truly representative. Filtration may also be advantageous.

Step 2

Carefully apply the sample so that it sits over the black spot between the four
alignment spots, (see Figure: 4).

Take care not to introduce bubbles into the sample.

After the plunger is depressed, lift the pipette up carefully so as not to drag the
droplet off-target.

Step 3

Observe the position and shape of the drop; it should resemble the one shown
in Figure: 6a rather than 6b. A noticeably spread-out sample indicates the target
area may be contaminated and requires cleaning (see below). Gently lower the
sampling head.

Figure 4

Figure 5

Figure 6a

Figure 6b

Return light path

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5061-049 REV 1.4

Step 4

If a program has been selected and the Auto-Read function is set to On (see
section 2.6), the sample reading process will begin automatically – a reference
scan will be taken first, followed by the measurement scan.

If the Auto-Read function is set to Off, use the 0A/100%T and

buttons to take
the reference and measurement scans respectively. Note: It is not recommended
to raise and lower the head repeatedly to take multiple measurements of one
sample, this can cause the droplet to disperse. If repeat measurements are
required use the button

Step 5

If you wish to keep the sample it can be recovered after the reading has been
taken using a pipette.

Step 6

After taking the reading, both the top and bottom plates should be cleaned
by wiping away the sample using a soft lint-free tissue. Wipe the bottom plate
towards you and the top plate upwards to avoid contaminating the return light
path to the rear (see previous figure 6a).

It may be necessary to remove sticky or dried-on residues. Water or a dilute (2%)
detergent solution should suffice. After using detergent, the plates should be wiped
a second time with either water or Isopropanol (2-propanol).

3.2. Sample Plate replacement

If after careful cleaning the droplet shape is still unsatisfactory, or if either of
the plates becomes damaged, or there is deterioration of the hydrophobic
coating, then it will be necessary to replace them as a pair. The glass plates are
permanently fixed within plastic housings which clip to the sample handling
module. Replacements are available from your supplier.

Exchanging the sample plates is simple (see Figures: 8a and 8b). They are available
from your supplier under part number 28-9244-06

1. Remove the top sample plate. Put one thumb at the base of the sample plate

and with the other hand gently lever down the tab at the top of the plate.

2. Replace the top sample plate. Insert the bottom tab and hold this in place with

one thumb and then gently push the tab at the top until the plate clicks into
place. Once in position, two silver pins will be visible either side of the top sensor.

3. Remove the bottom sample plate. Gently lever the plate upwards using the

thumb tab at the front of the plate and then pull it towards you to remove it
completely.

4. Replace the bottom sample plate. Gently insert the tabs at the back of the plate

as far as they will go and then push down the front of the sample plate so the
tabs underneath are fully pushed in.

5. Always calibrate the system after replacing the sample plates.

Figure: 7a

28955301.

Locate the screw above the bottom sample plate. Turn counter clockwise to loosen.
Remove the bottom sample plate. Gently lever the plate upwards using the thumb tab
at the front of the plate and then pull it towards you to remove it completely.

Replace the bottom sample plate. Gently insert the tabs at the back of the plate as far
as they will go and then push down the front of the sample plate so the tabs underneath
are fully pushed in. Insert screw back in, turn clockwise to tighten.

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5061-049 REV 1.4

3.3. Cleaning and general maintenance

Before cleaning the case of the instrument, switch off the instrument and
disconnect the power cord.

Clean all external surfaces using a soft damp cloth. A mild liquid detergent may be
used to remove stubborn marks.

3.4. Return for repair

The responsibility for decontamination of the instrument lies with the customer.
The case may be cleaned with mild detergent or an alcohol such as ethanol or
Isopropanol.

Examination or repair of returned instruments cannot be undertaken unless
they are accompanied by a decontamination certificate signed by a responsible
person.

Figure: 8a

Figure: 8b

3.3. Cleaning and general maintenance

Before cleaning the case of the instrument, switch off the instrument and
disconnect the power cord.

Clean all external surfaces using a soft damp cloth. A mild liquid detergent may be
used to remove stubborn marks.

3.4. Return for repair

The responsibility for decontamination of the instrument lies with the customer.
The case may be cleaned with mild detergent or an alcohol such as ethanol or
Isopropanol.

Examination or repair of returned instruments cannot be undertaken unless
they are accompanied by a decontamination certificate signed by a responsible
person.

This could be of the following form:-

3.5. Lamp replacement

The xenon lamp should not need replacement until used for several years. In the
unlikely event that it does fail, this should be undertaken by a service engineer
from your supplier.

3.6. Replacing the printer paper

Spare paper for the printer (20 rolls) is available from your supplier under part

number 28918226

Step 1 Keep the power on. Lift off the paper cover.
Step 2 Lock the platen (horizontal position) and feed the paper into the slot.

The drive will engage automatically and take up the paper.

Step 3 It may help to release the platen lock (turn flat green catch clockwise) and

turn the green knob manually.

Step 4 Replace the cover.

Figure: 8a

Figure: 8b

Please contact technical support at [e-mail] for assistance.

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4. LIFE SCIENCE

4.1. File system

The Life Science Screen is organized into six sub-folders as shown below:

Folder Keypad Number Application Function

1 DNA Concentration and purity check for DNA samples
2 RNA Concentration and purity check for RNA samples
3 Oligo Concentration and purity check for Oligonucleotide samples
4 Tm Calculation DNA melting point calculator
5 Cy Dye Labeling efficiency measurement
6 Protein Folder containing methods for protein determination

Pressing 6 enters the Protein sub-folder which contains various standard methods
for protein determination:-

4.2. Nucleic Acids

4.2.1. Theory

•Nucleicacidscanbequantifiedat260nmbecauseatthiswavelengththere

is a clearly defined peak maximum. A 50 µg/ml DNA solution, a 40 µg/ml RNA
solution and 33 µg/ml solution of a typical synthetic Oligonucleotide all have an
optical density of 1.0 A in a 10 mm pathlength cell. These factors (50, 40 and 33
respectively) can be inserted into the formula (1) below, although they do vary
with base composition and this can be calculated more precisely if the base
sequence is known.

(1) Concentration = Abs260 * Factor (where * means “multiplied by”)

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5061-049 REV 1.4

•NanoVuePluswilldefaulttofactors50fordoublestrandedDNA,40forRNA

and 33 for single stranded DNA and Oligonucleotides. It also allows manual
compensation for dilution - aided by a dilution calculator.

•Nucleicacidsextractedfromcellsareaccompaniedbyproteinandextensive

purification is required to remove the protein impurity. The 260/280 nm
Absorbance ratio gives an indication of purity, however it is only an indication
and not a definitive assessment. Pure DNA and RNA preparations have expected
ratios of 1.7–1.9 and ≥ 2.0 respectively. Deviations from this indicate the
presence of impurity in the sample, but care must be taken in the interpretation
of results.

•The260nmreadingistakennearthetopofabroadpeakintheAbsorbance

spectrum for nucleic acids, whereas the 280 nm reading naturally occurs on
a steep slope, where small changes in wavelength will result in large changes
in Absorbance. Consequently, small variations in wavelength accuracy have
a much larger effect at 280 nm than at 260 nm. It follows therefore that
the 260/280 ratio is susceptible to this effect and users are warned that
spectrophotometers of different designs may give slightly different ratios.

•Inpractice,concentrationalsoaffectsthe260/280ratioastheindividual

readings approach the instrument’s detection limit. If a solution is too dilute, the
280nm reading shows a greater proportional interference from background and
as the divisor becomes smaller, has a disproportionate effect on the final result.

It is advisable to ensure that the Abs260 value is greater than 0.1 A for accurate
measurements.

•AnelevatedAbsorbanceat230nmcanalsoindicatethepresenceofimpurities.

230 nm is near the Absorbance maximum of peptide bonds and may also
indicate interference from common buffers such as Tris and EDTA. When
measuring RNA samples, the 260/230 ratio should be > 2.0. A ratio lower than
this is generally indicative of contamination with guanidinium thiocyanate, a
reagent commonly used in RNA purification, which absorbs over the
230–260 nm range. A wavelength scan of the nucleic acid is particularly useful
for RNA samples.

•NanoVuePlusdisplaystheindividualAbsorbancevaluesandthe260/280and

260/230 ratios on the left side of the screen and attention should be given to
these as well as the final result on the right hand side.

Use of Background Correction

•BackgroundCorrectionatawavelengthwellapartfromthenucleicacidor

protein peaks is often used to compensate for the effects of background

Absorbance.Theprocedurecanadjustfortheeffectsofturbidity,stray

particulates and high-Absorbance buffer solutions.

•NanoVuePlususesBackgroundCorrectionat320nmbydefaultonallLife

Science Applications, particularly nucleic acid measurements. It is particularly
recommended since very small samples are particularly susceptible to stray
particulates. The Background function toggles On and Off with either left/right
arrows from the relevant page.

•Ifitisused,therewillbedifferentresultsfromthosewhenunused,because

Abs320 is subtracted from Abs260 and Abs280 prior to use in equations:
Concentration = (Abs260 – Abs320) * Factor
Abs ratio 260/280 = (Abs260 – Abs320) / (Abs280 –Abs320)
Abs ratio 260/230 = (Abs260– Abs320) / (Abs230 –Abs320)

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Note:

•TheAbsorbancemaximumnear260nmandAbsorbanceminimumnear230nm.

•Theflatpeaknear260nmandsteepslopeat280nm.

•ThereisverylittleAbsorbanceat320nm.

4.2.2. DNA measurement

The procedure is as follows:

Step 1

Press 1 to select DNA mode.

Step 2

Select Pathlength using the left and right arrows. Options are 0.5 mm, 0.2 mm or
Automatic. Press the down arrow.

Step 3 (Dilution Factor known)
Enter the Dilution Factor using the keypad numbers (range 1.00 to 9999). Use the C
button to backspace and clear the last digit entered.

OR

Step 3 (calculate Dilution Factor)
Press

 to enter the Dilution Factor Screen (see second parameter screen to

the left).

Enter the Volume of the sample using the keypad numbers (range 0.01 to 9999).
Press the down arrow.

Enter the volume of the Diluent using the keypad numbers (range 0.01 to 9999).

Press OK

to calculate the Dilution Factor and return to the Parameters Screen

OR Press Cancel to cancel the selections and return to the Parameters Screen.

Step 4

Background Correction at 320 nm will be on. It may be turned off with the left or
right arrows. Press the down arrow.

Step 5

Select the Units of measurement using the left and right arrows. Options: µg/ml,
ng/µl, µg/µl. Press the down arrow.

Step 6

Enter the Factor using the keypad numbers. The default value is 50, the range is

0.01 to 9999.

Step 7
Press OK

to enter the Results Screen and begin making measurements.

Typical spectral scan of a Nucleic Acid:-

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OR

Esc

to return to the Life Science Screen.

Results Screen

Step 8

Pipette on the reference sample and lower the sampling head. If Auto-Read is
off, press the 0A/100%T key. This will be used for all subsequent samples until
changed. If QA is switched on the sample will need to be replaced and the
0A/100% T key pressed again.

Step 9

Clean the top and bottom plates, pipette on the sample and lower the sampling
head. If Auto-Read is off, press

. This measures at the selected wavelengths
and displays the results. The ratio of the Absorbance values at wavelengths 1 and
2 are calculated. The Concentration is based on the Absorbance at wavelength 1.
Repeat step 9 for all samples.

Press

Esc

to return to the Life Science Screen.

Press

 to display available Options which are described below.

Options (select using keypad numbers)

1. Return to Parameters Screen (step 1 above).

2. Print result via selected method.

3. Toggle Graph on/off. The graph shows a Wavescan plot across the range

220 nm to 320 nm with cursors denoting 230, 260, 280 and (if Background
Correction selected) 320 nm.

7. Sample Number – add a prefix to the sample number and reset the

incrementing number to the desired value.

8. Save Method – use the alpha-numeric keys to enter a name for the method and

press Save

.

9. Auto-Print – toggles Auto-Print on/off.

Exit Options by pressing

Esc

or wait.

4.2.3. RNA measurement

The procedure is as follows:

Step 1

Press 2 to select RNA mode.

Step 2

Select Pathlength using the left and right arrows. Options are 0.5 mm, 0.2 mm or
Automatic. Press the down arrow.

Step 3 (Dilution Factor known)

Enter the Dilution Factor using the keypad numbers (range 1.00 to 9999). Use the
C button to backspace and clear the last digit entered.

OR

Step 3 (calculate Dilution Factor)

Press

 to enter the Dilution Factor Screen (see second image to the left).

Enter the Volume of the sample using the keypad numbers (range 0.01 to 9999).

Press the down arrow.

Enter the volume of the Diluent using the keypad numbers (range 0.01 to 9999).

Press OK

to calculate the Dilution Factor and return to the Parameters Screen.

OR Press

Esc

to cancel the selections and return to the Parameters Screen.

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Step 4

Select whether the Background Correction at 320 nm is to be used or not with the
left and right arrows.

Press the down arrow.

Step 5

Select the Units of measurement using the left and right arrows. Options: µg/ml,
ng/µl, µg/µl.

Press the down arrow.

Step 6

Enter the Factor using the keypad numbers. The default value is 40, the range is

0.01 to 9999.

Step 7

Press OK

to enter the Results Screen and start making measurements.

OR

Esc

to return to the Life Science Screen.

Results Screen

Step 8

Pipette on the reference sample and lower the sampling head. If Auto-Read is
off, press the 0A/100%T key. This will be used for all subsequent samples until
changed. If QA is switched on the sample will need to be replaced and the
0A/100% T key pressed again.

Step 9

Clean the top and bottom plates, pipette on the sample and lower the sampling
head. If Auto-Read is off, press

. This measures at the selected wavelengths

and displays the results. The ratio of the Absorbance values at wavelengths 1 and
2 are calculated. The Concentration is based on the Absorbance at wavelength 1.

Repeat step 9 for all samples.

Press

Esc

to return to the Life Science Screen.

Press

 to display available Options which are described below.

Options (select using keypad numbers)

1. Return to Parameters Screen (step 1 above).

2. Print result via selected method.

3. Toggle Graph on/off. The graph shows a Wavescan plot across the range

220 nm to 320 nm with cursors denoting 230, 260, 280 and (if Background
Correction selected) 320 nm.

7. Sample Number – add a prefix to the sample number and reset the
incrementing number to the desired value.

8. Save Method – use the alpha-numeric keys to enter a name for the method and
press Save

.

9. Auto-Print – toggles Auto-Print on/off.

Exit Options by pressing

Esc

, or wait.

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4.2.4. Oligonucleotide measurement

The procedure is as follows:

Step 1

Press 3 to select Oligo mode.

Step 2

Select Pathlength using the left and right arrows. Options are 0.5 mm, 0.2 mm or
Automatic. Press the down arrow.

Step 3 (Dilution Factor known)
Enter the Dilution Factor using the keypad numbers (range 1.00 to 9999). Use the C
button to backspace and clear the last digit entered.

OR

Step 3 (calculate Dilution Factor)
Press

 to enter the Dilution Factor Screen.

Enter the Volume of the sample using the keypad numbers (range 0.01 to 9999).

Press the down arrow.

Enter the volume of the Diluent using the keypad numbers (range 0.01 to 9999).

Press OK

to calculate the Dilution Factor and return to the Parameters Screen.

OR Press

Esc

to cancel the selections and return to the Parameters Screen.

Step 4

Select whether the Background Correction is to be used or not with the left and
right arrows.

Press the down arrow.

Step 5

Select the Units of measurement using the left and right arrows. Options: µg/ml,
ng/µl, µg/µl and pmol/µl. If pmol/µl is selected the factor changes to a selection
table denoting the ratios of the 4 bases in the structure.

Press the down arrow.

Step 6 (Units not pmol/µl)
Enter the factor using the keypad numbers. The default value is 33, the range is

0.01 to 9999.

OR

Step 6 (Units pmol/µl)
Enter the proportions of bases present using the keypad numbers and up and
down arrows to move between boxes. The default is 10 for each, the range is 0 to

9999.

Step 7

Press OK

to enter the Results Screen and start making measurements

OR

Esc

to return to the Life Science Screen.

Results Screen

Step 8

Pipette on the reference sample and lower the sampling head. If Auto-Read is
off, press the 0A/100%T key. This will be used for all subsequent samples until
changed. If QA is switched on the sample will need to be replaced and the
0A/100% T key pressed again.

Step 9

Clean the top and bottom plates, pipette on the sample and lower the sampling
head. If Auto-Read is off, press

. This measures at the selected wavelengths

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5061-049 REV 1.4

and displays the results. The ratio of the Absorbance values at wavelengths 1 and
2 are calculated. The Concentration is based on the Absorbance at wavelength 1.

Repeat step 9 for all samples.

Press

Esc

to return to the Life Science Screen.

Press



to display available Options which are described below.

Options (select using keypad numbers)

1. Return to Parameters Screen (step 1 above).

2. Print result via selected method.

3. Toggle Graph on/off. The graph shows a Wavescan plot across the range

220 nm to 320 nm with cursors denoting 230, 260, 280 and (if Background
Correction is selected) 320 nm.

7. Sample Number – add a prefix to the sample number and reset the
incrementing number to the desired value.

8. Save Method – use the alpha-numeric keys to enter a name for the method and
press Save

.

9. Auto-Print – toggles Auto-Print on/off.

Exit options by pressing

Esc

, or wait.

4.2.5. Tm Calculation

This utility calculates the theoretical melting point from the base sequence of a
primer. It is done using nearest neighbor thermodynamic data for each base in the
nucleotide chain in relation to its neighbor (Breslauer et al, Proc.Natl. Acad. Sci. USA
Vol 83, p3746 1986). The data obtained are useful both in the characterization of
oligonucleotides and in calculating Tm for primers used in PCR experiments.

The ACGT/U sequence entered into the utility is used to calculate the theoretical
T

m

, the theoretical Absorbance (Absorption units/mmol) and the conversion factor
(mg/ml). This is because the stability of a bent and twisted sequence of bases such
as an oligonucleotide is dependent on the actual base sequence. The calculated

thermodynamicinteractionsbetweenadjacentbasepairshavebeenshownto

correlate well with experimental observations.
This utility uses matrices of known, published thermodynamic values and extinction

coefficients to calculate T

m

and the theoretical Absorbance/factor respectively of an

entered base sequence.

Tm

This is calculated using the equation:-

Tm = ∆H × 100

- 273.15 +16.6 log [salt]

∆S + (1.987 × loge(c/4 +53.0822)

where ∆H and ∆S are the enthalpy and entropy values summed from

respective 2 × 4 × 4 nearest neighbor matrices

c is the Primer concentration of oligonucleotide (pmoles/ml) in the

calculated Tm or the measured concentration in measured Tm.
In the latter case concentration is obtained from the equation:

c = Abs(260 nm) × Calculated factor × pathlength multiplier × 10 000
MW

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Calculated factor and MW are defined below

[salt] is the buffer molarity plus total molarity of salts in the hybridization

solution (moles/l)

Weights for ∆Sareindexedbyadjacentpairedbases.

A similar equation applies to weights for ∆H,againindexedbyadjacentbases.

Note that bivalent salts may need normalizing using a multiplying factor of 100
because of their greater binding power.

Theoretical Absorbance

The Theoretical Absorbance is based on a calculation as follows:

Foreachadjacentpairofbases(nearestneighbors)anextinctioncoefficientweight

is accumulated using a 4 × 4 table (one for either DNA or RNA). This total weight
is doubled and then for each internal base a counterweight is subtracted using
another 1 × 4 table. The end bases are excluded from the latter summation.

That is:

Total Extinction Coefficient E = ∑ (2 × aTable[base_type][base(n)][base(n+1)])

- ∑ (tTable[base_type][base(n]])

Conversion factor

The Conversion Factor is given by = Molecular weight

ABCDE

∑ E

ABCDE

where
E

ABCDE

= [ 2 × (EAB +EBC +ECD +EDE) –EB –EC –ED ]

•Themolecularweight(MW)ofaDNAoligonucleotideiscalculatedfrom:

MW (g/mole) = [(dA × 312.2) + (dC × 288.2) + (dG × 328.2) + (dT × 303.2.)] + [(MW
counter-ion) × (length of oligo in bases)]

(for RNA oligonucleotide, (dT × 303.2) is replaced by (dU × 298.2)

 TheMWcalculatedusingthisequationmustbeadjustedforthecontributionof

the atoms at the 5’ and 3’ ends of the oligo.

For phosphorylated oligos: add: [17 + (2 × MW of the counter-ion)]
For non-phosphorylated oligos: subtract: [61 + (MW of the counter-ion)]

The MW (g/mole) of the most common oligo counter-ions are;
Na (sodium) 23.0
K (potassium) 39.1
TEA (triethylammonium) 102.2
Other Defaults to 1.0 (H) Variable 0.1–999.9 in
next option box.

Calculated molecular weight: a weight is added for each base looked up from a
table. The weight of the counter ion is added for every base from a small table for
the known ions. If phosphorylated, then the system adds 17.0 plus two counter ions
otherwise it subtracts 61.0 and one ion.

TheoreticalAbsorbance:foreachadjacentpairofbases(nearestneighbors)a

weight is accumulated using a table. For each internal base a weight is subtracted
using another table. Separate tables are used for DNA and RNA.

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Calculated factor:thisisjustthecalculatedmolecularweightdividedbythe

theoretical Absorbance.

The procedure is as follows:

Step 1

Press 4 to enter the Tm Calculation Parameters Screen.

Step 2
Select the Base Type: DNA or RNA.

Press the down arrow.

Step 3
Select whether the sample is Phosphorylated or not (Yes or No).

Press the down arrow.

Step 4
Enter the Primer Conc. using the keypad numbers (range 0.000 to 99.9) in
pmole/ml.

Press the down arrow.

Step 5
Enter the Buffer Molarity (buffer molarity + total molarity of salt in moles/l) using
the keypad numbers (range 0.000 to 10).

Press the down arrow.

Step 6
Select the Counter Ion: Na, K, TEA or Other.

Step 7 (if Other selected, see the previous section)

Enter the molecular weight (Other MW) of the Counter Ion used.

Step 8
Press Next

to select these parameters and go on to the Base Sequence

Screen

OR

Press Cancel

Esc

to return to the Life Science Screen.

Step 9
Select the Pathlength of the sample cells. Options are 0.5 mm, 0.2 mm or
Automatic.

Step 10
Enter the known Base Sequence triplets using the number keys: 1 for A, 3 for C, 4
for G and 6 for T or U. Note that a comma is added after each triplet to improve
readability

Step 11
Press OK

to select these parameters and start to measure Tm

OR

Press Cancel

Esc

to return to the Parameters Screen.

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5061-049 REV 1.4

Results Screen

Step 12

Pipette on the reference sample and lower the sampling head. If Auto-Read is
off, press the 0A/100%T key. This will be used for all subsequent samples until
changed. If QA is switched on the sample will need to be replaced and the
0A/100% T key pressed again.

Step 13
Clean the top and bottom plates, pipette on the sample and lower the sampling
head. If Auto-Read is off, press

. The unit measures the Absorbance and uses

this information to calculate the Measured Tm.

Repeat step 13 for all samples.

Press

Esc

to return to the Life Science Screen.

Press



to display available Options which are described below.

Options (select using keypad numbers)

1. Return to Parameters Screen (step 1 above).

2. Print result via selected method.

7. Sample Number – add a prefix to the sample number and reset the

incrementing number to the desired value.

8. Save Method – use the alpha-numeric keys to enter a name for the method and
press Save

.

9. Auto-Print – toggles Auto-Print on/off.

Exit options by pressing

Esc

, or wait.

4.2.6. CyDye measurement

Measurement of the labeling efficiency of fluorescently labeled DNA probes
before 2-color microarray hybridization ensures that there is sufficient amount of
each probe to give satisfactory hybridization signals. These data also provide an

opportunitytobalancetherelativeintensitiesofeachfluorescentdyebyadjusting

the concentration of each probe before hybridization. The DNA yield is measured
at 260nm while the incorporation of fluorescein, Cy3, Cy5 and other dyes are
measured at their absorption peaks. This method may also be useful for measuring
the yields and brightness of fluorescently labeled in-situ hybridization probes.

Equations used in the software are detailed below:

DNA Concentration
= (A

260

– A

Background

) * NucleicAcid Factor

DNA Quantity
= DNAConcentration * Volume

Dye Concentration
= ((A

Dye

- A

Background

) / ExtinctionCoefficient

Dye

) * PathlengthFactor * Dilution_Factor

* 10

6

Dye Quantity
= DyeConcentration * Volume

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Dye Incorporation
= DyeQuantity / DNAQuantity

Dye FOI
= DyeQuantity * (324.5 / DNAQuantity)

Step 1
Press 5 to enter the Cy Dye Parameter Screen from the Life Sciences screen.

Step 2
Either select the Dye Name of the dye being used by using the left and right
arrows, or select “Custom” if you wish to create a new entry. If a standard dye
is selected, press Next to move to the next screen (Step 7). If “Custom” has been
selected press the down arrow.

Step 3 (Custom only)
Enter the Wavelength of the dye peak Absorbance.

Press the down arrow.

Step 4 (Custom only)
Enter the dye Extinction Coefficient.

Press the down arrow.

Step 5 (Custom only)
Enter the dye 260 nm Correction factor (if known), otherwise set at zero.

Step 6 (Custom only)
Enter a name for the dye to identify it correctly.

To complete the entry press Next

to enter the second dye detail entry screen.

OR

Press Cancel

Esc

to return to the Life Science Screen.

Step 7
If there is no second dye select None, otherwise proceed as for Dye 1.

Press Next

to complete data entry and enter the Parameter screen

OR

Press Cancel

Esc

to return to the Dye 1 data entry screen.

Step 8

If the concentration is known select the appropriate Pathlength, otherwise leave it
as Automatic

Step 9
Unless there are complicating absorbencies at the Background wavelength, leave
this as On.

Step 10
If using standard dyes, the Background Wavelength is set automatically, but
if there are interferences at this default wavelength, the user can select a
wavelength where the background is at or close to the reference sample. If no
such wavelength is available set the previous box Background Correction to Off.
This will give rise to noisier results though