BIO RAD SmartSpec 3000 User manual

SmartSpec™3000
Spectrophotometer
Instruction Manual
Catalog Number
170-2501
For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)

Table of Contents
Page
Section 1 General Safety Information........................................................................1
1.1 Caution Symbol..........................................................................................................1
1.2 Precautions .................................................................................................................1
Section 2 Product Description.....................................................................................1
2.1 Packing List................................................................................................................1
2.2 Identification of System Components........................................................................2
Section 3 Installation....................................................................................................4
3.1 Environmental Requirements.....................................................................................4
3.2 Power On and Initial Display.....................................................................................4
Section 4 Introduction..................................................................................................5
4.1 Setup...........................................................................................................................7
4.2 Serial Port Functions................................................................................................10
Section 5 Key Pad Descriptions.................................................................................11
Section 6 Quick Guide................................................................................................13
Section 7 Operating Instructions..............................................................................15
Section 8 Description of Assays.................................................................................23
Section 9 Troubleshooting..........................................................................................46
9.1 Troubleshooting Table.............................................................................................46
Section 10 Maintenance ...............................................................................................47
10.1 Cleaning Up a Fluid Spill.........................................................................................47
10.2 Cleaning Quartz Cuvettes ........................................................................................47
10.3 Changing Thermal Printer Paper .............................................................................48
Section 11 Specifications ..............................................................................................49
11.1 Specifications ...........................................................................................................49
11.2 Calculations..............................................................................................................49
Section 12 System, Accessories, and Reagents for SmartSpec
Spectrophotometer.....................................................................................51
12.1 Product Information .................................................................................................51
12.2 Bio-Rad Protein Assays ...........................................................................................51
12.3 Cuvette Selection Guide...........................................................................................52
12.4 Selecting the Appropriate Cuvette...........................................................................52
12.5 PC Serial Connection Instructions...........................................................................53
For technical service call your local Bio-Rad office. In the U.S., call 1-800-4BIORAD
(1-800-424-6723).

Warranty
The SmartSpec 3000 Spectrophotometer and accessories are warranted against defects
in materials and workmanship for one year. If any defects occur in the instruments or acces-
sories during this warranty period, Bio-Rad Laboratories will repair or replace the defective
parts at its discretion without charge. The following defects, however, are specifically exclud-
ed:
1. Defects caused by improper operation.
2. Repair or modification done by anyone other than Bio-Rad Laboratories or an authorized
agent.
3. Damage caused by substituting alternative parts.
4. Use of fittings or spare parts supplied by anyone other than Bio-Rad Laboratories.
5. Damage caused by accident or misuse.
6. Damage caused by disaster.
7. Corrosion caused by improper solvent†or sample.
For any inquiry or request for repair service, contact Bio-Rad Laboratories. Inform Bio-
Rad of the model and serial number of your instrument.
Important: This Bio-Rad instrument is designed and certified to meet EN61010-1*
safety standards. Certified products are safe to use when operated in accordance with the
instrument manual. This instrument should no be modified or altered in any way.
Alteration of this instrument will:
Void the manufacturer's warranty
Void the EN61010-1 safety certification
Create a potential safety hazard
Bio-Rad Laboratories is not responsible for any injury or damage caused by the use of this
instrument for purposes other than those for which it is intended, or by modifications of the
instrument not performed by Bio-Rad Laboratories or an authorized agent.
†SmartSpec 3000 Spectrophotometer is not compatible with chlorinated hydrocarbons (e.g., chloroform), aromatic hydrocarbons (e.g.,
toluene, benzene) or acetone. Use of organic solvents voids all warranties.
*EN61010-1 is an internationally accepted electrical safety standard for laboratory instruments.

Section 1
General Safety Information
This instrument is intended for laboratory use only.
This product conforms to the "Class A" standards for electromagnetic emissions intend-
ed for laboratory equipment applications. It is possible that emissions from this product may
interfere with some sensitive appliances when placed nearby or in the same circuit as those
applications. The user should be aware of this potential and take appropriate measures to
avoid interference.
1.1 Caution Symbol
Read the manual before using the SmartSpec 3000 Spectrophotometer. For technical
assistance, contact your local Bio-Rad office or, in the U.S., call technical services at
1-800-4BIORAD (1-800-424-6723).
Definition of Symbols
Caution (refer to accompanying documents)
1.2 Precautions
1. Do not use near flammable materials.
2. Always inspect the SmartSpec 3000 for damaged components before use.
3. Always connect the system to the correct AC power source.
4. Always connect the correct instrument (printer or computer only) via the serial port
connector.
5. Do not pour liquid into the sample chamber. Thorough clean-up is needed after each spill
(Section 10.1).
6. Do not place objects on the SmartSpec 3000.
7. The SmartSpec 3000 has no customer-serviceable components, do not open the unit.
Section 2
Product Description
2.1 Packing List
The SmartSpec 3000 Spectrophotometer (catalog number 170-2501) is shipped with the
following components. Upon receiving your instrument, please check that all items listed
below were shipped. If any items are missing or damaged, contact your local Bio-Rad office.
Item Quantity
Instruction manual 1
Warranty card (please complete and return) 1
SmartSpec 3000 Spectrophotometer 1
Power cable 1
Cuvette spring 1
Thermal paper roll 1
1
!!
!
!!

2.2 Identification of System Components
Fig. 1. View of the SmartSpec 3000 Spectrophotometer.
Instrument
1. Keypad interface Change menu and select options by pressing these
buttons. Press a single button to perform action
described on the menu display.
2. Display 2 x 24 alphanumeric backlit LCD display. Displays
various menus and options available.
3. Sample compartment door Lift door to insert cuvette into the cuvette chamber.
4. Integrated thermal printer Built-in alphanumeric/graphic printer. Press print
button to record results immediately.
5. Printer paper compartment Remove door to install new roll of printer paper
door
Fig. 2. Rear view of the SmartSpec 3000 Spectrophotometer.
6. Fan Guard Exhaust air leaves cabinet through this opening.
7. Universal Power Input Module Appropriate power cord is connected to the power
and ON/OFF Switch input module. Instrument power switch.
8. Serial Output Port RS-232 connection to serial printer or computer.
2
1
3
7
86
4
5
2

Fig. 3. View of the SmartSpec 3000 sample compartment.
9. Cuvette chamber Insert cuvette into the cuvette chamber. Insure that the
cuvette is placed in the proper orientation with respect to
the light path.
10. Cuvette storage compartment Storage space for cuvette.
11. Cuvette spring Aligns cuvette. Accommodates various cuvette
configurations.
12. Light path Light path axis.
Fig. 4. View of the SmartSpec 3000 integrated thermal printer paper with door removed.
13. Thermal printer paper Roll of thermal printer paper.
14. Printer paper shaft
3
10
9
12
11
13
14

Section 3
Installation
3.1 Environmental Requirements
To insure correct operation and stable performance over an extended period of time, install
the SmartSpec 3000 in a location which meets the following conditions:
• Room temperature between 20 and 35 °C. Not recommended for cold room use (4 °C).
• Not exposed to direct sun light.
• Not subject to direct or continuous vibration.
• Not subject to intense magnetic or electromagnetic fields.
• Relative humidity between 0–95%.
• Area free from corrosive gases or other corrosive substances.
• Area with very little dust or other airborne particles.
• Allow a 10 cm minimum space around the instrument for proper air flow.
3.2 Power On and Initial Display
1. Power up SmartSpec. When you turn on SmartSpec 3000, it will go through a self-
diagnostic routine during which it tests the lamp, optical components, diode array, power
supply and other components. If a problem is detected, SmartSpec 3000 will alert you to call
Bio-Rad Technical Support. If boot up is successful and no errors are detected, the Home
screens will be displayed.
alternating with
4
CHOOSE ANY
SMARTSPEC ASSAY
04:23:11 PM SmartSpec
Fri Apr 23, 1999

5
Section 4
Introduction
UV/Visible Spectroscopy is based on the absorption of light as a function of wavelength.
All spectrophotometers have a light source that generates light of specific wavelengths. The
SmartSpec 3000 uses a xenon flash lamp as its light source. Xenon flash lamps have several
advantages over the conventional light sources in that they reduce warm up time, do not heat
up the instrument and have a lamp life of 10 to 15 years. Additionally, xenon flash lamps
emit both UV and visible wavelengths, in contrast to deuterium lamps which emit only UV
wavelengths. In the SmartSpec 3000, the xenon flash lamp pulses light only when measuring
absorbance of a sample.
Fig. 5. A symbolic representation of the SmartSpec 3000 optics module.
The path of light in the SmartSpec 3000 is as follows:
Light from the xenon flash lamp
Lens
Sample
Slit
Concave mirror
Grating
Concave mirror
Photodiode array detector
The advantages of using a grating as a dispersion device are that:
1. They eliminate nonlinear dispersion.
2. Are not temperature sensitive.
Photodiode arrays have several advantages:
1. They acquire spectral data simultaneously without mechanically moving parts;
2. Have excellent wavelength reproducibility and,
3. A wide dynamic range.
It is also important to note here that in an array, several photodiode detectors are arranged
on a silicon crystal. The added benefit of an array is the ability to do side-by-side readings, thus
increasing speed.
Source
Lens Sample Slit Grating Detector
Concave
Mirror Concave
Mirror

The Lambert-Beer’s law identifies the relation between sample concentration and the
intensity of transmitted light. Absorbances and % transmittances are defined by the following
equations:
% Transmittance (%T)
%T = It/I0*100=10
-εcl * 100
Absorbance or optical density (A or O.D.)
A = log 10 1/T = log 10 I0/It= log10 10εcl =εcl
Where I0= Intensity of incident light
It= Intensity of transmitted light
ε= Molar extinction coefficient (liter/mole/cm)
c = Sample concentration (mol/liter)
l = Optical pathlength
Fig. 6. Lambert-Beer's law.
A = log 1/T A = εcl
Fig. 7. Relationship between Transmittance and Concentration and Absorbance and
Concentration.
6
Transmittance
100
Absorbance
Concentration Concentration
Sample concentration C
ll
ll
Incident light loTransmitted light lt=l
o10-εεcll
ll

4.1 Setup
When the SmartSpec 3000 is not carrying out an assay, you can enter the Setup routine
by pressing Setup. To proceed through Setup without changing the current setting, press
Enter. Most of the current settings can be changed by pressing Select until you see your
desired choice; sometimes you have to use the number pad to change an entry. In either case,
press Enter after the selection is made. When you have gone all the way through Setup,
SmartSpec 3000 will automatically return to Home screen. You can exit Setup at any time by
pressing Cancel. When you exit Setup, all changes will be saved.
In Setup you can specify the following:
1. Pathlength
2. Background compensation for DNA/RNA measurements.
3. Default type of nucleic acid.
4. Default method to determine DNA and RNA oligonucleotide molar extinction
coefficient and molecular weight.
5. Default number of sample replicates.
6. New operator name.
7. Current operator.
8. Serial port export.
9. Current time.
10. Current date.
11. Time display mode.
12. Date display mode.
1. Pathlength. The pathlength describes the distance that the light travels as it passes through
the sample, and is the same as the width of the cuvette. By using a cuvette with a shorter
pathlength, you can measure the absorbance of more concentrated solutions. The
relationship between absorbance, path length and sample concentration is described by
Beer’s law where
A=εbc
A is the absorbance, εthe molar extinction coefficient in liter/mole/cm, b the pathlength
in cm and c the concentration in moles/liter.
Pressing Select once will change the screen to
As you continue pressing Select, the choices change to 2 mm and then 1 mm.
7
Select Pathlength:
>=Help 10 mm
Select Pathlength:
>=Help 5 mm

2. Background compensation. When using the DNA/RNA assay, you can choose to have
SmartSpec 3000 automatically subtract background absorbance readings collected at 320
nm from the A260 and A280 measurements. If the background subtraction option is
invoked, then the A320 is subtracted from the A260 and A280 readings before these
measurements are displayed and before they are used in the calculation of nucleic acid
concentration. The default condition is to subtract the A320 background. You can toggle
background subtraction on and off by pressing Select.
3. Default type of Nucleic Acid. SmartSpec 3000 can convert absorbance reading of nucleic
acid solutions into concentration. The conversion factor is determined by the type of
nucleic acid being measured. You can select the default type of nucleic acid in Setup, so
that when you press DNA/RNA, this type of nucleic acid will be shown first on the
display. You can choose another type of nucleic acid at any time by pressing Select. The
choices are double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA,DNA
oligo and RNA oligo. The factory default choice is dsDNA.
4. Default method to determine oligo molar extinction coefficient and molecular weight.
SmartSpec 3000 uses the molecular weight and molar extinction coefficients of
oligonucleotides to convert the absorbance readings into mass (µg/ml) and molar (pmol/µl)
concentrations. You can input the molecular weight and molar extinction coefficient
directly if you know them, otherwise SmartSpec 3000 will use one of three methods to
estimate them for you: Length, Composition or Sequence. The simplest method to
estimate either the molecular weight or the molar extinction coefficient is by the length
of the oligonucleotide. This method assumes an equivalent distribution of all four
nucleotides among the oligo. More accurate estimates can be calculated if you specify
the composition (i.e., the number of A’s, C’s, G’s and T’s or U’s) of the oligo and the best
estimate of molar extinction coefficient is calculated with the sequence of the
oligonucleotide.
Press Select to change the default to Composition (Compstn) and press Select again to
set the default to Sequence.
5. Default number of sample replicates. SmartSpec 3000 has the ability to accept samples
(and standards) in replicate groups and to calculate a mean and standard deviation for
each sample (or standard) replicate group. The factory default number of sample
replicates is 1, but you can change the number of replicates to any number between 1 and 9.
Use the keypad to choose the number of sample replicates and press Enter or Setup when
finished. During most assays you can change the number of sample replicates by
choosing Options from the Ready Screen.
8
Substract A320 Background
for DNA/RNA? Yes >=Help
Select default type of
DNA/RNA: dsDNA >=Help
Select default method of
find oligo conc: length
Enter default number of
sample replicates:

6. New Operator. When SmartSpec 3000 prints a report, it includes the name of the oper-
ator (if specified). You can add a new operators name to the list of operators in Setup.
Press Select to toggle from No to Yes and the screen will change to
The keyboard will automatically switch to the alpha mode and you can use the keys to
enter a name with as many as eight characters. To enter an Ain the first space, press the 2
key once. To enter a B, press the 2 key twice, and to enter a C, press it three times. To
move from the first space to the second space, press the right arrow key. When the last
letter of the name has been specified, press Enter. If you want to mix numerals with letters,
you can toggle back and forth between the alpha mode and number mode by pressing
Alpha. When the LED on the Alpha key is lit, then the keyboard is in alpha mode. You can
enter a space by pressing the right arrow key twice.
7. Current operator. Press Select until the desired name is displayed on the screen and
then press Enter. This name will be printed on the final report.
8. Serial port export. When this feature is activated, absorbance data and concentration data
are sent out through the serial port in all assays. Otherwise only the Scan and Kinetic
assay data can be sent out the serial port.
• To export data through the serial port, press Select to toggle from No to Yes and
press Enter. If this feature is active, then absorbance data from the DNA/RNA,
Protein, OD600 and λassays will be exported when you press Print. Those data
will be printed at the same time.
9. Current time. The time will be displayed in the present format.
If the displayed time is incorrect, press Select and then Enter.
• Use the number pad to enter the hour and press Enter, then enter the minutes and press
Enter again. After setting the time, press Select to change from AM to PM if you are
using the 12-hour format. Press Enter when complete.
10. Current date. The date will be displayed in the present format.
If the date is not correct, press Select to toggle from Yes to No and Enter.
9
Want to add the name of
a new operator? No
Enter new operator's
name: (8 char):
Current time: 1:12 PM
is this OK? Yes
Do you want to send data
through serial port? No
Enter new time. Select
AM or PM: ____ : ____ AM
Current date: (MM/DD/YY)
11/25/98 Is this OK? Yes
Enter new date in format
MM/DD/YY: ___/___/YY

• Use the number pad to input the new date. Press Enter to move to the next field. Press
Select to set the day of the week. Press Enter when you are finished.
11. Time convention. You may choose to print time in 12-hour or 24-hour mode. Toggle
between the choices by pressing Select. and then press Enter.
Press Select for
12. Date convention. You can choose to print the date in either MM/DD/YY or DD-MM-YY
mode. Toggle between the choices by pressing Select and then press Enter.
Press Select for
13. Exit Setup. After going through the entire Setup menu, the display will return to the
Home screen. All changes will be saved. You can also exit Setup at any time by pressing
Cancel and all changes up to that point will be saved.
4.2 Serial Port Functions
The SmartSpec 3000 has the ability to communicate with an external printer or
computer via the serial port located at the back of the instrument. The SmartSpec 3000 sends
data to these peripherals as ASCII text. When connecting the SmartSpec 3000 to an external
printer or computer, a "standard serial" cable should be used. The correct pin-out for this cable
is described in the table below. Check the printer or computer for the correct serial port
connector end (male or female).
DB25F Connector Signal and DB25M Connector
to SmartSpec 3000 3000 (male) Direction to serial device
2 TX--> 3
3 <--RX 2
4 RTS--> 5
5 <--CTS 4
7 Common 7
20 DTR--> 20
(other pins are open in SmartSpec 3000)
The serial port parameters are: 9600 baud rate, 8 data bits, no parity, 1 stop, and no flow
control. These parameters are pre-set in the unit. When using a serial printer set the printer to
the serial port parameters specified above. Please note that the data is sent from the SmartSpec
3000 to a computer or serial printer as an extended hex character. If your computer or printer
is not set to accept this character please refer to the instrument’s instruction manual for correct
settings. (For detailed instructions see Section 12.5.)
10
Select date display mode
MM/DD/YY e.g. 11/25/98
Select date display mode
DD-MM-YY e.g. 25-11-98
Select time display mode
24-hr e.g. 13:12
Select date display mode
DD-MM-YY e.g. 25-11-98

Section 5
Keypad Description
Print SmartSpec 3000 will print data on the current sample, a
user-selectable range of samples or all samples in the
current assay.
Abs SmartSpec 3000 will automatically display the
absorbance reading of the current sample and the
wavelength being read. If more than one wavelength is
being read, press Abs to toggle among the different
wavelengths. If you are reading one of a replicate group,
you can choose to see the absorbance of the current
replicate or you can see the mean and standard
deviation of all members of the replicate group by first
pressing Abs.
Conc The Conc key is used in the DNA Oligo and RNA Oligo
assays to toggle the display of concentration from units
of µg/ml to pmoles/µl. This key has no function for the
Scan, Kinetics and λassays.
A260:A280 SmartSpec 3000 will calculate and display the ratio of
A260 to A280. This is a method of approximating the
purity of nucleic acid preparations; pure preparations of
DNA have ratios of about 1.8 and pure RNA preparations
have a ratio of about 2.0. If you are using background
compensation (in Setup), then the ratio is calculated after
the background reading of A320 is subtracted from the
A260 and A280.
Dilution Factor The dilution factor is used as a final multiplier in calcu-
lation of concentration from absorbance data. The
default dilution factor is 1.0 and the dilution factor is
reset to 1.0 each time that one of the Assay buttons is
pressed.
Setup SmartSpec 3000 will enter the setup routine where you
can specify the pathlength, defaults for certain options,
time and date convention and time and date. You exit
Setup either by proceeding all the way through the rou-
tine, or by pressing Cancel at any time. When you exit
the Setup routine, all changes are saved.
Assay Buttons The six assay buttons are listed below. Press one of these
to begin an assay with SmartSpec 3000. When any assay
button is pushed, SmartSpec 3000 resets the dilution fac-
tor to 1.0, the sample number to 1, and checks the data
buffer for unprinted data.
DNA/RNA SmartSpec 3000 will automatically collect absorbance
data at 260, 280 and 320 nm, and use those readings to
calculate nucleic acid concentration. The calculation of
concentration depends on the type of nucleic acid
specified.
11

Protein SmartSpec 3000 will automatically read one or more
wavelengths depending on the type of assay specified
(Bradford, Lowry, BCA or UV Protein) or the user can
specify a wavelength. SmartSpec 3000 will convert
absorbance data to concentration by use of a standard
curve, except for the case of UV Protein in which the
absorbance readings are multiplied by user-selectable
constants. The standard curve can be one recalled from
memory, or a new one constructed by SmartSpec 3000.
Scan SmartSpec 3000 will collect and print the absorbance
of a sample over a user-defined range of wavelengths.
An optional background wavelength may be specified
and its absorbance will be subtracted from all
absorbances in the scan range before the scan is
displayed. SmartSpec 3000 will identify and quantitate
the points of maximum and minimum absorbance over
the scanned range. These data can be printed or
downloaded via the serial port to an external computer.
Kinetics SmartSpec 3000 will collect data from a single cuvette
at fixed periodic intervals over a certain time period.
Both the interval of data collection and length of data
collection are specified by the user. An optional
background wavelength may be specified. The kinetic
readings may be printed or exported to external software
for analysis.
OD600 SmartSpec 3000 will collect absorbance data at 600 nm.
This assay is used for monitoring the growth of bacterial
cultures. The user may specify a conversion factor which
is multiplied by the dilution factor and the absorbance to
calculate the concentration in cells per ml.
λλSmartSpec 3000 will collect data at up to three different
user-specified wavelengths. An optional background
wavelength may also be specified and its absorbance
will be subtracted from the other wavelengths before
they are reported.
Read Blank SmartSpec 3000 will zero the detector in preparation for
reading samples. It is important to zero the instrument at
each new assay. Insert a cuvette that contains all assay
components except the analyte and press Read Blank.
Read Sample SmartSpec 3000 will collect absorbance data from the
cuvette in the instrument.
Select Used to toggle between or among choices. Once the
choice is made, its selection is confirmed by pressing
Enter.
Enter Indicates to SmartSpec 3000 that a choice has been
made.
12

Cancel Cancels the last entry or causes an exit from Setup.
When editing sequence information, press Cancel to
delete a base. When Options are displayed, press Cancel
to return to the assay.
and Used to access other menus or screens. Also used to
move the cursor.
Paper Feed Feeds paper through the printer.
Alpha The Alpha key allows the user to toggle between alpha
and numeric input for naming stored standard curves or
naming operators. When in alpha mode, an LED is
illuminated on the key. To type an alphabetic character
when in the alpha mode, press one of the number keys
in the left or center columns. To type an A, press the 1
key once; to type a B, press the 1key twice, and to type
aC, press the 1key three times. These keys also enable
the user to enter sequence data. Sequence data are input
using the last column of keys on the number pad: to
type an A, press the 3key; to type a C, press the 6key;
to type a G, press the 9key and to type a Tor U, press
the exp key.
Number pad Used to input numeric values for SmartSpec 3000
calculations. The exp key is used for making entries in
exponential notation, and when it is pressed, an εεis
displayed by SmartSpec 3000. It refers to the base 10
and not to the base of the natural logarithm.
Section 6
Quick Guide
1. Power up the instrument. It will go through system status check and alert you if there are
problems.
2. Press one of the Assay buttons.
A. DNA/RNA.
i. Choose type of nucleic acid
a. dsDNA, ssDNA or RNA: accept or modify the conversion factor
b. DNA oligo or RNA oligo: input molar extinction coefficient and molecular
weight; otherwise choose method for SmartSpec 3000 to estimate these values.
ii. Choose whether to subtract background and, if so, specify background
wavelength.
B. Protein
i. Choose type of assay.
a. Bradford. Measure absorbance at 595 nm.
b. Lowry. Measure absorbance at 750 nm.
c. BCA. Measure absorbance at 562 nm.
d. UV. Measure absorbance at 260, 280, and 320 nm.
e. Other. User specifies the wavelength to read.
><
13

ii. Choose standard curve option.
a. Create a new standard curve.
b. Recall a standard curve from memory.
c. No standard curve. SmartSpec 3000 will not be able to convert absorbance
to concentration.
C. Scan
i. Set upper and lower limits of scan.
ii. Choose whether to subtract background and, if so, specify background
wavelength.
iii. Choose fast or slow scan.
iv. For the fast scan, choose number of successive scans.
D. Kinetic
i. Choose wavelength to read.
ii. Choose duration of data collection.
iii. Choose interval between successive readings.
iv. Choose whether to subtract background and, if so, specify background
wavelength.
E. OD600
i. Accept or modify the conversion factor.
F. λ
i. Choose number of wavelengths to read.
ii. Specify the wavelengths to read.
iii. Choose whether to subtract background and, if so, specify background
wavelength.
3. If your dilution factor is not 1.0, set the dilution factor.
4. Zero the instrument. Place a cuvette containing the blank solution into SmartSpec 3000
and press Read Blank.
5. Collect absorbance data. Place cuvette containing sample solution into SmartSpec 3000
and press Read Sample. Continue collecting absorbance data until all samples are read.
6. Absorbance and/or concentration data are displayed automatically as they are collected.
If there are absorbance data at other wavelengths, press Abs to see them. DNA or RNA
oligo concentrations are available in units of µg/ml and pmole/µl; press Conc to toggle
between them. If absorbance or concentration data area displayed, press Enter to return
the Ready screen, or simply put the next sample cuvette into SmartSpec 3000 and press
Read Sample.
7. After last sample, press left arrow key to exit assay.
8. Save the standard curve (if you made a new one) and print the full report.
14

Section 7
Operating Instructions
Note: It is Good Laboratory Practice (GLP) to use the Smart Spec 3000 spectrophotometer
to measure absorbances in the range of 0.100 to 1.500 AU.
1. Choose an assay. SmartSpec 3000 has six different assay keys in the second row of
buttons. When you press one of them, it signals SmartSpec 3000 to clear its memory of
absorbance readings from the previous assay, to reset the sample number to 1, and to
reset the dilution factor to 1. Before the data buffer is cleared, SmartSpec 3000 will offer
to print any unprinted data. All of the assays collect absorbance data and some of the
assays are designed to convert those readings into concentrations or other useful
information. The user interface of each assay is described in detail in the next section.
The available assays are:
A. DNA/RNA. In this assay, SmartSpec 3000 will automatically collect absorbance
readings at 260, 280 and 320 nm. If you choose to subtract the background at 320 nm
(in Setup), then the A320 reading is subtracted from the A260 and A280 readings
before the A260 and A280 absorbances are displayed or used in any calculations of
nucleic acid concentration or in calculation of the A260:A280 ratio.
Calculations. Nucleic acid concentrations of dsDNA, ssDNA and RNA are
determined by multiplying the A260 absorbance reading by a mass/absorbtion
conversion factor and by the dilution factor, or by multiplying the difference between
the A260 and A320 by the conversion and dilution factors.
Concentration = A260 * Conversion Factor * Dilution Factor
or
Concentration = (A260–A320) * Conversion Factor * Dilution Factor
SmartSpec 3000 is programmed with default conversion factors for dsDNA, ssDNA
and RNA. These factors can be modified at any time.
Species A260 of 1.000
dsDNA 50.0 µg/ml
ssDNA 37.0 µg/ml
RNA 40.0 µg/ml
DNA and RNA oligonucleotide concentrations are reported in two different ways: by
mass (µg/ml) and moles (pmol/µl); you can toggle between the two values by pressing
Conc. In order to make these determinations, SmartSpec 3000 requires the molecular
weight of the oligo and either the molar extinction coefficient or the mass/absorbance
conversion factor (as shown in the table above for dsDNA, etc.). Frequently the molar
extinction coefficient and/or the mass/absorbance conversion factor is provided when
you order an oligonucleotide from a custom synthesis laboratory. However, if you
don’t know this factor, SmartSpec 3000 can estimate them for you in one of three dif-
ferent ways. In order of increasing accuracy, by length of the oligo, by composition of
the oligo, (i.e., how many A’s, C’s, G’s and T/U’s) or by the primary sequence of the
oligo. See Section 11.2 for a complete explanation of the calculations.
B. Protein. The Protein function will automatically select the proper wavelength for
the Bradford (595 nm), Lowry (750 nm), BCA (562 nm) or UV Protein (260, 280,
and 320 mm) assays, or you can select a different wavelength for another assay. The
Bradford, Lowry and BCA are all well-characterized colorimetric assays for
protein concentration. The UV protein assay can determine protein concentration in a
nucleic acid background.
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The SmartSpec 3000 Bradford, Lowry, BCA and Other Protein assays use standard
curves created by measuring the absorbances of solutions of known concentration to
determine the concentration of unknown samples. If you do not want to construct a
new standard curve, you may recall one from the on-board memory. SmartSpec
3000 has the capability of storing up to ten standard curves in memory. The stored
curves are numbered automatically from 0 to 9, or you can overwrite the number.
Stored curves may also be assigned alphabetic names using the Alpha function of the
number pad.
You also have the option of declining construction or recall of a standard curve, but
without the standard curve, absorbance readings cannot be converted to
concentrations.
The UV Protein assay, developed to find protein concentration in a nucleic acid
background, as in a crude cell extract, calculates protein concentration by the
equation
Concentration = C1 * (A280 –A
320)–C2*(A 260 –A 320)
where C1 and C2 are constants. If you select the UV Protein assay, SmartSpec 3000
will first show you the current values for C1 and C2 and ask you to accept or
modify them. The preset values for C1 and C2 are 1.55 and 0.76 respectively.
Standard Curve Construction. SmartSpec 3000 will calculate a mean and
standard deviation for each standard replicate group (if replicates are specified) and
then use those data to fit a linear standard curve. The instrument will report the slope
and intercept of the standard curve and the square of the correlation coefficient for
the curve (r2).
The correlation coefficient (r) provides an indication of how well the linear
polynomial regression model fits the data. In effect, the correlation coefficient
is the square root of the proportion of explained variation to total variation of
the regression.
r = (explained variation / total variation)1/2
The amount of explained variation increases as the goodness of fit improves. In a
perfect fit, all of the variation is explained, the explained variation equals the total
variation, and the ratio of the explained variation to total variation becomes 1.000.
Or, more simply, the closer the correlation coefficient is to 1.000, the better the fit
of the regression, and the better the estimate of concentration.
Storing Standard Curves. As many as ten standard curves may be stored in
SmartSpec 3000’s on-board memory. These may be recalled for use in determining
concentrations in subsequent assays. A new curve will be assigned a number from
0 to 9 by the firmware and you can then give it an optional name of up to eight
characters in length.
Printing Standard Curves. If you choose to print a full report at the end of an
assay, the standard curve will be printed along with the information about the curve.
This will include the concentrations and absorbances of the standard and the slope,
intercept and coefficient of the curve.
You can choose to print the same information about a standard curve when it is first
created or when it is recalled from memory.
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Limitations of the methods. The linear regression, when fit properly, generally
produces reliable results at the extremes of the range of the standard curve. Results
calculated from mean absorbance data outside the range of the standard curve may
be unreliable. (For the purposes of this discussion, the range of the standard curve is
defined as the absorbance range between the highest and lowest mean absorbances
of the standards used to form the standard curve.) Calculated results that are
determined from mean absorbances outside the range of the standard curve are
extrapolations, and should be treated as such when determining the final results of
your analysis.
The firmware will not be able to give a unique concentration for a given absorbance
if the calibration data are not monotonic (i.e., two standards of differing concentration
have the same absorbance reading).
C. Scan. SmartSpec 3000 can read any subset of the interval between 200 nm and 800 nm
and then print the scan; the interval can be further subdivided into as many as four
regions, and for each region the local minimum and maximum are identified. You
may select a background wavelength and its absorbance will be subtracted from the
absorbance at all other wavelengths in the interval of the scan. There are two
scanning speeds: fast and slow. If you choose a slow scan, you will get the best
data, but will take several minutes to complete, depending on the sample and the
range being read. You may not specify more than one repetition for slow
scanning. Scan data may be downloaded to an external computer via the serial port,
or may be printed by SmartSpec 3000.
D. Kinetic. SmartSpec 3000 can collect absorbance readings at periodic intervals for
a specified length of time so that you can collect kinetic data. You may specify a
wavelength for background subtraction; background absorbances are collected at the
same interval and are subtracted from the reading wavelength. You may then print
out the data and/or download the data to another program for analysis. Permissible
reading intervals range from 2 seconds to 999 seconds and data may be collected
for as long as 9999 seconds.
E. OD600. This function is useful for monitoring growth of bacterial cultures.
SmartSpec 3000 can also convert the absorbance reading into a concentration with
units of cells/ml if provided with a conversion factor. The conversion factor is
multiplied by the absorbance reading and the dilution factor to estimate the
concentration of cells in cells per ml. For example, if for certain E. coli an OD of 1.0
indicates a concentration of 5x108cells/ml, then the conversion factor would be
5x108. On the SmartSpec 3000 5x108is entered in exponential notation using the
exp key (press 5, exp, 8 and Enter) and displayed as 5e8. On the display, e refers
to the base 10 and not to the base of the natural logarithm.
F. λλ. The wavelength function allows you to choose up to three wavelengths for
measurement and any other wavelength for optional background subtraction. It is a
rapid and simple way to collect absorbance data without constructing standard curves
or otherwise calculating concentrations.
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