BIO RAD SmartSpec 3000 Instruction Manual
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BIO RAD SmartSpec 3000 Instruction Manual

BIO RAD SmartSpec 3000 Instruction Manual

Spectrophotometer
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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)

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Summary of Contents for BIO RAD SmartSpec 3000

  • Page 1 ™ 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)

  • Page 2: Table Of Contents

    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).
  • Page 3 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.

  • Page 4: General Safety Information

    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...

  • Page 5: Identification Of System Components

    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.
  • Page 6 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...

  • Page 7: Installation

    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.

  • Page 8: Introduction

    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.
  • Page 9 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) -εcl %T = I * 100 = 10 * 100 Absorbance or optical density (A or O.D.) εcl = εcl A = log...

  • Page 10: Setup

    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;...
  • Page 11 (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.
  • Page 12 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. Want to add the name of a new operator?

  • Page 13: 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"...

  • Page 14: Key Pad Descriptions

    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. SmartSpec 3000 will automatically display the absorbance reading of the current sample and the wavelength being read.
  • Page 15 λ λ SmartSpec 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.

  • Page 16: Quick Guide

    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.
  • Page 17 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.

  • Page 18: Operating Instructions

    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.
  • Page 19 ) – C2 * (A – A 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.
  • Page 20 (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.
  • Page 21 If the default number of sample replicates has been set to 2–9 (in Setup), then SmartSpec 3000 will ask if any of the samples in this assay are to be read in replicate. The default answer is No.
  • Page 22 3 >=Option • Use the number keys to input the number of replicates and press Enter. The absorbance data for the first replicate will be collected and then SmartSpec 3000 will direct you to insert the second replicate Insert...
  • Page 23 Press Print to begin printing the scan. If you do not want to print the scan, press Cancel. From this screen, you can press the left arrow key to select nm scale and direct SmartSpec 3000 to print only a subset of the data collected. Begin scanning at ____ nm...
  • Page 24 • Now press Select and the negative sign will be displayed in front of 0.500. Min AU scale: +/- -0.500 Max AU scale: • Press Enter again and now define the upper limit. The upper limit must be a positive number.
  • Page 25 Ready to Read Absorbance <=Exit Assay >=Options After the data are downloaded SmartSpec 3000 will reset for the next kinetic data col- lection. From the Ready screen, you can change any of the parameters for the kinetic data collection. E. OD600. Immediately after the absorbance data are collected, they are displayed along with the cellular concentration.

  • Page 26: Description Of Assays

    <=Exit Assay >=Options SmartSpec 3000 will prompt you to save a newly created standard curve and then prompt you to print a complete report of absorbances, concentration and A260:A280 ratios. The report will also show means and standard deviations for each standard or unknown sample replicate group.
  • Page 27 If you choose DNA oligo or RNA oligo, you must provide the molecular weight of the oligo and either the molar extinction coefficient or the mass/absorbance conversion factor. If you don’t know them, then SmartSpec 3000 can estimate them for you. Do you know oligo molar extnctn coefficient? •...
  • Page 28 C’s. After entering the number of T’s (or U’s for RNA oligos), press Enter. SmartSpec 3000 will calculate the molecular weight and display it along with the molar extinction coefficient.
  • Page 29 Mole Ext Coef=xxxxxxx NW=xxxxx >=Continue • Press the right arrow key to continue the assay. SmartSpec 3000 is now ready to collect absorbance measurements and to convert those to concentrations. Ready to read absorbance <=Exit Assay >=Options iii.
  • Page 30 Use the number keys to input the length of the oligo and press Enter. You can enter a length of up to 99 nucleotides. After you press Enter, SmartSpec 3000 will calculate and display the extinction coefficient and molecular weight.
  • Page 31 • Press the right arrow to continue the assay. SmartSpec 3000 will now be ready to measure the absorbance of your oligonucleotide and to convert those absorbance data into concentrations of µg/ml and pmol/µl. Mol ext coef= __________ _________ >=Continue Protein Assay 1.
  • Page 32 If you do not want to make a new standard curve, press Select to toggle from Yes to No and press Enter. A. If you choose not to create a new standard curve. SmartSpec 3000 will offer you the opportunity to recall a standard curve from memory.
  • Page 33 <=Back >=Continue • Press the right arrow key to return to the assay. SmartSpec 3000 is now ready to collect absorbance data and to covert the absorbances into concentrations using the standard curve recalled from memory. Ready to read absorbance <=Exit Assay...
  • Page 34 Select concentration units: none Next SmartSpec 3000 will ask if any of the standards are to be read in replicate. Select concentration units: µg/ml • If you have prepared any of your standards in replicate, press Select to toggle from No to Yes and press Enter.
  • Page 35 • Press the right arrow key to return to the assay. Once all the standards are read, SmartSpec 3000 will calculate a standard curve. Skip ahead to C. ii. If there are standard replicates. If you indicate that one or more of the standards are read in replicate, then you will be asked if there is the same number of replicates for each standard.
  • Page 36 #1. After all replicates of standard #1 are read, then you will be asked for the concentration of standard #2, and then directed to input the replicates of standard #2. SmartSpec 3000 will continue in this manner until all replicates of all standards are measured.
  • Page 37 After all replicates of standard #1 are measured, SmartSpec 3000 asks for the concentration of standard #2 and directs you to put the replicates into SmartSpec 3000 one at a time until all are read. Enter concentration of Standard #2:...
  • Page 38 After all replicates of the first standard are read, then SmartSpec 3000 will prompt for the number of replicates of the second standard and then for the concentration of the second standard. This will continue until the last replicate of the last standard is read.
  • Page 39 After all replicates of standard #1 are measured, SmartSpec 3000 asks for the number of replicates of standard #2 and then the concentration of standard #2 before prompting you to insert each of the replicates of standard #2. Enter number replicates for Standard #2 (1–9): __...
  • Page 40 C. Editing standard curve data. After the absorbance of the last replicate of the last standard has been read, SmartSpec 3000 will offer you a chance to edit the data set before constructing the standard curve. You can re-read a standard or any replicate of a standard group, or you can edit the concentration information for any standard or standard replicate group.
  • Page 41 SmartSpec 3000 will now direct you to insert blank (if you chose that option above) and the to insert the specified standard. When the absorbance data have been collected, SmartSpec 3000 will offer you an opportunity to continue re-reading other samples.
  • Page 42 If the slope or intercept has a value greater than 9999 or less than 0.0001, it will be shown in exponential notation. In all cases with SmartSpec 3000, e represents the base 10, not the base of the natural logarithm. For example, 100,000 would be represented as 1.000 e 05 and 0.00001 would be represented as 1.000 e-05.
  • Page 43 When you rescale the X axis, you must input the new maximum X value and press Enter. A new maximum Y value is automatically calculated and the graph is plotted. conc=150.0000 Enter new max: When you rescale the Y axis, you must input the new maximum Y value and press Enter. A new maximum X value is automatically calculated and the graph is plotted.
  • Page 44 256, 257, 258, 259, and 260 and plot this average at 258 nm. This is available through the Options menu. When you press Scan, SmartSpec 3000 will prompt for upper and lower limits of the scan. Begin scanning at ____ nm End scanning at ____ nm •...
  • Page 45 Use the number keys to input the number of successive scans and press Enter. SmartSpec 3000 will now display the Ready screen. If you want to invoke the smoothing option or the subregion option, choose Options from the Ready screen.
  • Page 46 <=Exit Assay >=Options After the kinetic data are collected, SmartSpec 3000 will reset for the next scan and then display the Ready screen. If you want to change any of the kinetic parameters, you can do that without exiting the assay if you choose Options from the Ready screen.
  • Page 47 Use the number keys and the exp key to input the new conversion factor and press Enter. SmartSpec 3000 is now ready to collect OD600 readings. For best results, you should first zero the SmartSpec 3000 by inserting a cuvette with media (without cells) and pressing Read Blank.
  • Page 48 λ λ This assay allows you to collect absorbance data from up to three different wavelengths simultaneously. No concentrations are calculated. When you press λ λ , SmartSpec 3000 will prompt you first for the number of wavelengths to read.

  • Page 49: Troubleshooting

    Paper loaded incorrectly. 2. Be sure the paper is installed in the proper orientation. Thermal print head/plate 3. Replace printer. Call Bio-Rad damaged. service. Keyboard buttons not Incomplete system 1. Power down unit/Restart unit. responding initialization during power up.

  • Page 50: Maintenance

    Problem Cause Solution Negative values/no No sample. 1. Add Sample to solution absorbancy Cuvette light path 2. Check cuvette window orientation incorrect. orientation. Wrong wavelength setting. 3. Check assay wavelength requirement. May have blanked the 4. Reblank the instrument using instrument using sample buffer or water.

  • Page 51: Changing Thermal Printer Paper

    Serrated edge paper tear Installing New Thermal Printer Paper Note: Use only Bio-Rad recommended thermal paper catalog number 170-2506. • Remove printer paper door cover by pushing down on the center of the door and sliding the door back away from the unit.

  • Page 52: Specifications

    Section 11 Specifications 11.1 Specifications Product Specifications SmartSpec 3000 Spectrophotometer Wavelength range: UV and Visible ranges 200 to 800 nm Wavelength accuracy (+/-) 2 nm Spectral bandwidth 5 nm Light source Xenon flash lamp Detector Photodiode array Number of cuvettes Size of cuvettes 100 µl, 700 µl, 1.5 ml, 3.5 ml...
  • Page 53 III. DNA and RNA Oligonucleotide Concentration: • Concentration = A/(εb) * 10 pmole/µl • Concentration = A/(εb) * MW * 10 µg/ml where A = absorbance, ε = molar extinction coefficient in liter/mole/cm, b = pathlength in cm and MW = molecular weight. For cuvettes with a 10 mm (1 cm ) pathlength, these equations simplify to: •...

  • Page 54: Product Information

    Catalog Number Product Description 500-0001 Bio-Rad Protein Assay Kit I, 450 standard assays, bovine gamma globulin standard, based on the method of Bradford 500-0002 Bio-Rad Protein Assay Kit II, 450 standard assays, bovine serum albumin standard, based on the method of Bradford...

  • Page 55: Cuvette Selection Guide

    10 mm 12.4 Selecting the Appropriate Cuvette The SmartSpec 3000 has 1 mm, 2 mm, 5 mm and 10 mm pathlengths. For most (99%) absorbance measurements you will be using 10 mm pathlength cuvettes. Cuvettes of shorter pathlength allow the measurement of concentrated samples without dilution. If you put a solution in a 10 mm pathlength cuvette and get a reading of 2.0 AU, you can use a 5 mm...

  • Page 56: Pc Serial Connection Instructions

    Echo typed characters locally Wrap lines that exceed terminal width 4. When the SmartSpec 3000 sends data, either when requested from Scan, or Kinetics, or when the user activates the data echo toggle in Setup, the data streams to the terminal...
  • Page 57 5. Capturing data in HyperTerminal and saving the data in a file: In the main HyperTerminal window menu, first click Transfer and then click Capture Text. Transfer Capture Text . . . [give file name and path] e.g. c:\program files\accessories\hyperterminal\capture.txt A dialog box with the default file name, file extension and file path will appear.
  • Page 58 Bio-Rad Laboratories Life Science Web site Group Also in: Australia Austria Belgium Ph. 09-385 55 11, Fx. 09-385 65 54 Brazil Denmark Finland France Ph. 01 47 95 69 65, Fx. 01 47 41 9133 Israel Italy Japan Korea The Netherlands Ph.

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