Hoefer DQ 200 - Genemco, Inc.
Contents
1. 48 DNA quantitation C Main bibliography ee 51 Ordering information 52 English O pi A Important user information Please read this entire manual to fully understand the safe and effective use of this product The exclamation mark within an equilateral triangle is intended to alert the user to the presence of important operating and maintenance instructions in the literature accompanying the instrument If you have any comments on this manual please send them to us at Hoefer Inc 953 Indiana Street San Francisco CA 94107 USA support hoeferinc com Hoefer Inc reserves the right to make changes in the specifications without prior notice Warranty and liability Hoefer Inc guarantees that the product delivered has been thoroughly tested to ensure that it meets its published specifications The warranty included in the conditions of delivery is valid only if the product has been installed and used according to the instructions supplied by Hoefer Inc Hoefer Inc shall in no event be liable for incidental or consequential damages including without limitation lost profits loss of income loss of business opportuni ties loss of use and other related exposures however caused arising from the faulty or incorrect use of the product O 2004 Hoefer Inc All rights reserved No part of this publication may be reproduced stored in2. 500 within 10 seconds because this solution photodegrades quickly Press lt ENTER gt The fluorometer will now display 500 for a 50 nM solution of AMU 2 0 ml of a 1 nM solution equals 2 pmol of 4MU Measure the fluorescence of the unknown sample Zero the instrument as in step 1 add 100 ul of the unknown sample mix thoroughly by pipetting with a disposable transfer pipette do not introduce bubbles Place cuvette into the well Close the lid and record sample fluorescence Repeat the measurement to verify that results are repro ducible Empty the cuvette between each measurement and rinse Drain the cuvette completely by blotting it while inverted on a paper towel e p27 Note The 200 nM standard should display about 2000 fluorescence units Worksheet B 4MU standard measurements Once the initial reference value can be reliably reproduced proceed to determine concentrations of unknown samples or determine assay linearity with standard dilution measurements Generating a standard dilution curve once every few weeks serves as a quality check on the standard a reli ability check on the instrument and a consistency check on technique Generate a standard dilution curve Example Determine measurements for the series of concen trations in Worksheet B below Add the appropri ate amount of carbonate stop buffer and place the cuvette in the cuvette well Close the lid and zero the instrument R
3. 3 gt Info 4 gt Diagnos Turning lamp on Zero first using a blank sample Blank gt sample Zero and re calib Re calib using lower value Failed Main menu option 3 gt Test Use the test menu to isolate the cause of a malfunction Press lt ESC gt to return to the Main Menu The four options are 1 gt The lt Data gt option displays voltage mV signals from the sample Sig and the lamp reference Ref 2 gt The lt Lamp gt option switches the lamp off if it is on or on if it is off 3 gt The lt Info gt option identifies the initial UV lamp reference signal mV the firmware version the PC board version the date of manufacture and the serial number 4 gt The lt Diagnos gt option initiates a comprehen sive system diagnostic routine The operator is required to press all 15 keys on the keypad and open and close the lid when prompted If a component is found faulty an error message displays the failure source Error and other messages This message reports that the lamp is being switched on Wait 15 minutes to allow the lamp to stabilize before taking measurements When this message shows the lamp was either inadvertently switched off or the auto shut function switched it off after an hour of no keypad activity The assay solution was not blanked Zero the instru ment place blank assay solution into the cuvette and press
4. connector The type of serial cable required depends on the type of device DTE or DCE that it is connected to The DQ 200 is configured as a DTE device so a connection to another DTE device requires a null modem serial cable If the data is delivered to a DCE device receives signals at pin 2 and transmits signals at pin 3 such as a printer then a regular serial cable is required DQ 200 RS232C signal and pin number assignments Pin 2 Transmit Pin 3 Receive Pin 5 Ground Other pins Not connected The DQ 200 requires these settings in the device receiving data Baud rate 1200 Data bits 8 Stop bit 1 Start bit al Parity None Fig 2 H 33258 binds to the minor groove of DNA When 365 nm light long UV excites this bound dye its fluorescence at 458 nm can be measured 2 Fluorometry principles and method overview Fluorescence measurement Bisbenzimide commonly known as Hoechst 33258 H 33258 dye exhibits changes in fluorescence characteristics in the presence of DNA that allow accurate DNA quantitation In the absence of DNA the excitation spectrum of H 33258 peaks at 356 nm and the emission spectrum peaks weakly at 492 nm When H 33258 binds to DNA these peaks shift to 365 nm ex and 458 nm em In the cuvette well the sample is exposed to filtered light 365 7 nm from a mercury lamp This light excites the DNA dye complex causing light that peaks at 458 nm to be emitted An emission filter in front of
5. control At this point the program enters the terminal mode To capture the text select Transfer Capture Text from the menu bar Fig 7 You will be prompted to enter or browse for the file to receive the captured data O Save the session parameters before exiting the program to save performing the setup steps the next time data is collected on the same computer e p17 e p18 4 DNA Quantitation This section includes guidelines for DNA assay solutions and standards the calibration protocol for the DNA standard supplied with the unit and a discussion of how to analyze measurements Refer to Appendix B for a discussion of other reference standards and a correction factor that can be applied if the base composition of the DNA standard used differs substantially from the DNA sample Guidelines for the H 33258 DNA assay Factors that affect the assay e The AT of a DNA sample affects H 33258 DNA fluorescence so it is important to use a standard similar to the sample under investigation The calf thymus DNA standard supplied can serve as a refer ence for most animal and plant DNA because it is double stranded highly polymerized and is approxi mately 42 GC 58 AT A different standard may be required for specific types of DNA such as bacterial DNA because the AT varies widely depending on the species as does the AT of some polymerase chain reaction PCR products Standards with a r
6. lt ZERO gt Blank value is higher than the sample value Zero using blank capillary assay solution and recalibrate The entered calibration value is too high Recalibrate with standard solution Use a lower factor if not using the actual standard concentration Diagnostic test failed Call the Hoefer Inc Technical Services Department e p15 Connection Description 2 xl DS New Connection Enter a name and choose an icon for the connection Name Icon Cancel Fig 4 Hyperterm opening screen epl6 DQ 200 communication with other devices Communication between the DQ 200 and another device such as a printer or an IBM compatible computer is limited to an ASCII dump No error checking such as CRC or protocols for attention such as ACK or NAK are available Also no XON or XOFF procedure is required To connect the communication facility plug the appropriate cable see page 5 into the DQ 200 serial port and the device Then select 2 gt Setup 3 gt Send to choose either Auto send which transfers each sample ID number and measurement automatically or Manual send which requires the operator to press lt SEND gt to transfer each reading to the device Software options If the data is transmitted to a computer it can be captured with terminal emulation software provided with most personal computer systems Standard installations of Microsoft Windows
7. a different AT Adjusted standard setting Csta 0 025 AT sta AT samp 1 The adjusted setting is the calibration to use for the standard at the Cstd concentration in ng ml Once adjusted the fluorometer reading will then give sample concentrations in ng ml For example if the standard and sample DNA have an AT of 40 and 50 respectively and the standard DNA concentration is 100 ng ml Adjusted standard setting 100 0 025 40 50 1 2 5 10 100 75 Thus setting the calibration to 75 for the 100 ng DNA standard will set the fluorometer to read directly in ng ml for the higher AT sample DNA e p49 Fig 12 Effect of AT on relative fluorescence DNA samples of Microccocus lysodeikticus AT 23 E coli strand B AT 50 calf thymus AT 58 Clostridium perfringens AT 69 and Poly dA Poly dT AT 100 all Sigma were dissolved in TNE buffer Initial concentrations were deter mined by A260 then diluted to either 100 ng ml or 1000 ng ml in assay buffer containing H 33258 The fluorometer was calibrated with calf thymus DNA at 100 ng ml then fluorescence readings were taken for each sample m DNA 1000 ng ml H 33258 1 0 pg ml DNA 100 ng ml H 33258 0 1 ug ml e p50 2000 1750 1000 ng ml 1500 1250 1000 Relative Fluorescence 750 f 500 250 A 0 20 40 60 80 100 DNA compostion AT An example using calf thymus DNA as a stand
8. a retrieval system or transmitted in any form by any means without permission in written form from the company Francais A Renseignements importants d utilization Pour une bonne compr hension et une utilisation en s curit maximale il convient de lire entierement ce manuel Dans la documentation qui accompagne l instrument un point d exclamation dans un triangle quilat ral a pour but d attirer l attention de l utilisateur sur des instructions importantes de fonctionnment ou de maintenance Tous vos commentaires sur ce manuel seront les bienvenus et veuillez les adresser Hoefer Inc 953 Indiana Street San Francisco CA 94107 USA support hoeferinc com Hoefer Inc se r serve le droit d effectuer des modifica tions de ces sp cifications sans aucun pr avis Garantie et responsabilit Hoefer Inc garantit l utilisateur que le produit livr a subi avec succ s tous les essais pr vus pour s assurer qu il est conforme aux sp cifications et normes en vigueur La garantie incluse dans les conditions de livrai son n est valable que si le produit a t install et utilis conform ment aux instructions fournies par Hoefer Inc La soci t Hoefer Inc ne sera en aucun cas responsable de tout dommage caus directement ou indirectement par toute utilisation incorrecte ou non approuv e du produit ou d coulant de cette utilisation y compris toute perte de b n fice ou de recettes
9. displays the Main Menu The lt ENTER gt key registers numeric values advances to the next screen or initiates a fluorescence measurement The lt SEND gt key sends sample ID num ber and displayed reading to the serial communication port 3 Operating instructions This section describes instrument operation For DNA quantitation protocols see Section 4 For GUS assays see Section 5 User interface The keypad is used to select setup options and to zero and calibrate the instrument The LCD display shows each current menu option DQ 200 Fluorometer The lt CALIB gt key establishes the fluo The lt ZERO gt key establishes the rescence level used to calculate the background fluorescence level to concentration of subsequent samples subtract from each sample p9 Hoefer DQ 200 System Diagnostic 1 gt Read 2 gt Setup 3 gt Test Important Allow 15 minutes for the lamp to stabilize before taking any measurements 1 gt Read 2 gt Setup 3 gt Test e plo Power up and program flow Turn the mains power switch beside the power cord receptacle to on I to activate a self diag nostic cycle which requires about 2 minutes This cycle identifies the manufacturer tests all circuits turns the UV lamp on and displays the Main Menu when the instrument is ready A series of screens report which components are being tested as the program runs through the system diagnostic
10. mM EDTA pH 7 4 H 33258 stock solution 100 ul 10X TNE 10 ml Distilled filtered water 90 ml Keep assay solution B at room temperature Prepare fresh daily Do not filter once dye is added Protocol Since the DNA fluorescence assay is based on a relative measurement of emitted light a calibration reference value must be established with a known DNA sample before the concentration of DNA in unknown samples can be determined Choose the standard concentration range low or high according to the expected DNA concentration and calibrate with this standard dilution One refer ence point is adequate to calibrate the instrument However generating a standard dilution curve assures assay linearity in the range of interest Generating a standard dilution curve once every few weeks serves as a quality check on the stan dard a reliability check on the instrument and a consistency check on technique Low range assay 10 to 500 ng ml final DNA concentration Assay solution A Standard 1 10 dilution to 100 ug ml of the 1 mg ml calf thymus DNA standard 2 ul of this solution mixed with 2 ml assay solution is a 100 ng ml standard solution High range assay 100 to 5000 ng ml final DNA concentration Assay solution B Standard Undiluted calf thymus DNA standard 1 mg ml 2 ul of this solution mixed with 2 ml assay solution is a 1000 ng ml standard solution The calf thymus standard is supplied in dry form Follow the instr
11. measuring DNA concentrations Spectrophotometer measurements of A260 and Azgo detect DNA RNA and protein DNA samples may appear to have higher concentrations by absorbance than by fluorescence due to the presence of contaminants For double stranded DNA the fluo rometric value is usually more accurate provided a clean DNA standard of known concentration is used Fluorescence of Iysates prepared without an alkaline EDTA pretreatment is reduced by 70 compared to Iysates with such pretreatment Alkaline conditions allow formation of complexes between DNA and the dye For a detailed protocol see Rymasz ewski et al 1990 Estimation of cellular DNA content in cell lysates suitable for RNA isolation Anal Biochem 188 91 96 Make sure to use a standard with a G C content very similar to the sample H 33258 binds preferentially to A T regions so G C content must be similar to ensure equivalent binding Use a ssDNA standard for ssDNA samples Single stranded DNA yields about 50 the fluorescence of an equal amount of double stranded DNA Plasmid DNA standards should have the same conformation as the sample Each form supercoiled relaxed circular or linear may have slightly different dye binding characteristics Fluorescence enhancement may result from high levels of deter gents Final SDS concentration should be below 0 01 and other detergents below 10 ug ml the final concentration of any detergent should be well be
12. mirror must fit flush with the top of the block 4 Install the emission filter so that the arrow points away from the block Install both seal rings Inspect the assembly If necessary wipe surfaces until clean Slide the assembled optical block into the instru ment and secure with the thumb screw e p33 Troubleshooting problem solution Always be sure to Fluorescence values drift Wide fluctuations in fluorescence values p34 Operate the unit in a location isolated from equipment that radiates high frequency electromagnetic interference Operate the unit away from direct sunlight Place the unit so that the back vents are not blocked Use no more than 2 ml of liquid in the cuvette and take care not to spill any liquid into the cuvette well Assay solutions must be at ambient temperature for consistent readings Fluorescence decreases as temperature increases Remove the cuvette from the well as soon as the measurement is taken to avoid heating and photobleaching destruction of the fluorescent compound by light Protect fluorescent reagents and samples from light to prevent photobleaching Take readings immediately after mixing in the cuvette Assay solutions must be at pH 7 4 Adjust the salt concentration For standard DNA extraction the concentration should be at least 200 mM NaCl in 1X TNE For crude cell lysates use 2 to 3 M NaCl in 1X TNE If air bubbles are prese
13. of 58 DNA standards with different AT are available and can be selected to match the characteristics of your sample A range of standards available from Sigma Chemical company are listed below Double stranded DNA source GC Sigma Chemical Co Ultra pure catalog number Calf thymus 42 D 4764 Clostridium perfringens 26 5 D 5139 E coli 50 D 4889 Human placenta 42 D 4642 Micrococcus luteus 72 D 5014 If measuring samples which differ significantly in AT from the selected DNA standard a modifica tion to the calibration protocol is needed As shown in Fig 12 fluorescence is a linear function of AT when the DNA concentration based on A260 is held constant This relationship was based on several DNA samples with AT ranging from 23 to 100 measured with the DQ 200 With a standard DNA concentration of 100 ng ml A260 0 0020 the slope of relative fluorescence versus AT is 2 5 while with DNA at 1000 ng ml the slope is 25 We noticed that poly dAT did not fit a linear plot as well as poly dA poly dT did suggesting that there is also a sequence dependent component of H 33258 binding Daxhelet B A et al Anal Biochem 179 401 403 1989 Because the AT effect and DNA concentration effect are both linear it is straightforward to calibrate the assay when the AT of the standard and sample differ Equation 1 gives an adjusted setting to use when a standard of one AT is used to calibrate the assay for sample DNA of
14. pll A N gt Instrument calibration E gt Measurement LCD message Prompt on If prompt on is selected the following steps are displayed Press the indicated key or press lt ENTER gt to continue Action required Place assay blank in well Press zero Computing zero Add calibration standard Press calib Enter std conc Calibrating Remove standard Place assay blank in well Concentration Press zero Computing zero Add unknown sample Concentration 1 gt Read 2 gt Calib esc gt Main Menu e pl Add only assay solution no standard to cuvette Always place cuvette into the well in the same orientation Close the lid Press lt ENTER gt The assay solution background is determined and subtracted Press lt ZERO gt Remove the cuvette and add the appropriate standard amount and concentration Mix by drawing the solution into a disposable transfer pipet several times Do not introduce bubbles into the solution Place cuvette into well close the lid and press lt ENTER gt Sets the instrument to display fluorescence units based on the standard Press lt CALIB gt Enter the standard concentration Display either ng ml or no units selected from the Setup menu Press lt ENTER gt Remove cuvette Press lt ENTER gt Drain and rinse cuvette Add only assay solution no sample to cuvette Place cuvette into the well always in the same orientat
15. readings exceed the display range gt 5000 dilute the sample until the reading is within the linear range of the assay as determined by a dilution curve If the unknown sample readings are very low add more sample Generate a standard concentration curve Generating a standard dilution curve verifies the linearity of the assay within a particular concen tration range The low range standard assay using assay solution A is linear for 10 ng ml to 500 ng ml final DNA concentration To maintain linearity above this range use a higher dye concentration assay solution B Example low range assay Calibrate the instrument with 100 ng ml DNA Determine the readings for the series of concentra tions in Worksheet A below Fill the cuvette with 2 ml of assay solution A place the cuvette in its original orientation in the cuvette well close the lid and zero the instrument remove the cuvette add the next volume of standard mix thoroughly replace the cuvette in the well and close the lid Record each reading Measure a second sample for each volume sample 2 and average the VO readings e p23 p24 Worksheet A Low range standard measurements DNA standard Reading Reading Avg reading y conc x vol ul sample1 sample2 samples 1 2 2 ng ml 0 0 100 2 200 4 300 6 400 8 500 10 100 ng ul DNA to be added to 2 ml assay solution A Standard curve volume calculations for the low range assay am
16. the photodetector allows only fluorescence at 460 15 nm to register With the appropriate dye concentra tion the measured fluorescence is a direct indicator of the DNA concentration The fluorescent glucuronidase GUS assay does not depend on the formation of a complex Instead measured fluorescence indicates the amount of reaction product 4 methylumbellif erone 4MU that has been released by the GUS hydrolysis of 4MU glucuronide The same excita tion and emission wavelengths apply however the shorter 365 nm wavelength light excites the fluo rescent 4MU moiety which then exhibits a peak emission at 455 nm Fluorometers measure fluorescence in relative rather than absolute units Thus after zeroing with a blank always begin an assay by calibrating the instrument to display the known concentration or a convenient multiple of a standard solution This relates the measured fluorescence of an unknown sample to the standard DNA quantitation is further discussed in Section 4 and the GUS assay is described in Section 5 For a current list of DQ 200 Application Notes see Ordering information Method overview All menu options are described in detail in the following sections Users familiar with fluorescent DNA assays and this instrument can refer to these steps or the laminated Quick Reference card for an abbreviated guide to measure the concentration of an unknown sample 0 Prepare the appropriate sta
17. when lt SEND gt is pressed 2 gt The Auto send option transmits this data automati cally after each measurement See pages 5 and 16 for printer connection guidelines After a brief pause the Setup menu is displayed e p13 5 gt Autoshut 6 gt ID 7 gt Language Lamp auto shut 1 gt 0ff 2 gt 0n Please enter ID number 0 1 gt Engl 2 gt Deutch 3 gt Franc 4 gt Espan EE ss e p14 Press lt 4 gt in the Setup menu for these additional options 1 gt Lamp auto shut off causes the lamp to stay on until the instrument is switched off That is the auto matic shut off is disabled 2 gt Lamp auto shut on causes the lamp to automati cally shut off after one hour of no keypad activity This option is recommended because it extends lamp life After a brief pause the Setup menu is displayed This option allows the operator to specify a starting sample ID number Each subsequent sample will then be assigned an ID number incremented by 1 from this starting point Input the starting point sample number Press lt ENTER gt To turn off sample numbering enter O After a brief pause the screen returns to options 5 7 in the Setup menu Press lt ESC gt for the Main Menu Enter the number corresponding to the desired language After a brief pause the screen returns to options 5 7 in the Setup menu Press lt ESC gt for the Main Menu 1 gt Data 2 gt Lamp
18. 0 and 5 64 Allen key Capillary tubes 9 ul glass 250 DQ130 9 Dye and standards Hoechst 33258 dye 100 mg DQ201 Calf thymus DNA standard 250 ug DQ202 4 methylumbelliferone standard 100 mg DQ203 Performance Validation Kit 1 DQ210 e p52 Clontech is a trademark of Becton Dickinson and Company Microsoft is a trademark of Microsoft Corporation Molecular Probes is a trademark of Molecular Probes Inc Research Organics is a trademark of Research Organics Inc Printed in the USA Hoefer Inc 953 Indiana Street San Francisco CA 94107 USA www hoeferinc com Hoefer
19. 198 20 Distilled water to 100 ml Store at 4 C away from light 4MU stock solution B 1 pM 4MU 10 ml 4MU stock solution A 10 ul Distilled water 10 ml Store at 4 C away from light Carbonate stop buffer 0 20 M 1000 ml Sodium carbonate anhydrous 21 28 MW 105 99 Double distilled water to 1000 ml Note If the reading exceeds the display range gt 5000 dilute the sample Protocol Since the 4MU fluorescence assay is based on a relative measurement of emitted light a calibration reference value must be established with a known sample before the activity of unknown samples can be determined The following steps assume the prompt is off If unfamiliar with the instrument select Prompt on by choosing 2 gt Setup from the Main Menu and then pressing lt 1 gt Since the concentration is not measured in ng ml select no units under 2 gt Setup from the Main Menu 2 gt Units 2 gt None Zero the instrument with assay solution If in place remove the glass cuvette from the cuvette well Add 1 9 ml of carbonate stop buffer to the cuvette If needed clean the sides of the cuvette with a low lint tissue Insert the cuvette always in the same orienta tion close the lid and press lt ZERO gt 2 Calibrate the unit Add 100 ul of stock solution B 1 uM 4MU mix by pipetting into a disposable transfer pipette several times close the lid and press lt CALIB gt Enter
20. E nanvai DQ 200 Hoefer DQ 200 fluorometer user manual um DQ200 IM Rev E0 06 04 A Ho e fe r Page finder 1 Fluorometer function and description UA PACKINGS seres eine a Specifications e cece eee Important information Instrument set up 2 Fluorometry principles and method overview Fluorescence measurement 6 Method overview 7 Important measurement notes 8 3 Operating instructions User interface 9 Power up and program flow 10 Main Mens er tell blas 10 Error and other messages 15 DQ 200 communications with other devices 16 4 DNA Quantitation Guidelines for H33258 DNA assay 18 SOON Scion ara aa een 20 Protocol 8 08 ensure 21 Generate a standard concentration curve 23 Analyze the results 25 5 Enzyme Activity Quantitation SOON cata a de 26 Protocol ci ee eee aan 27 Generate a standard concentration curve 28 6 Care and maintenance Cleaning ctra nd 30 Optical block disassembly 30 Optical block assembly 33 7 Troubleshooting eee 34 Appendices A Enzyme assay protocols B Glucuronidase 37 P G lactosidase un nen 43 B Effect of A T content on fluorescent
21. O Read sample fluorescence A typical time course assay result is shown in Fig 11 Fig 11 Typical time course assay results Relative Fluorescence uniuts Time min e p41 p42 Bibliography Jefferson R A Burgess S M and Hirsh D B glucuronidase from Escherichia coli as a gene fusion marker PNAS 83 8447 8451 1986 Jefferson R A Kavanagh T A and Bevan M W GUS fusions B glucuronidase as a sensitive and versatile gene fusion marker in higher plants EMBO J 6 3901 3907 1987 Jefferson R A Assaying chimeric genes in plants the GUS gene fusion system Plant Molecular Biology Reporter 5 387 405 1987 Jefferson R A The GUS reporter gene system Nature 342 837 838 1989 Segel I H Biochemical calculations John Wiley amp Sons New York 1976 Materials e DQ 200 Fluorometer glass cuvette e Microcentrifuge P galactosidase Assay Contributed by William A Braell Harvard Medical School Department of Biological Chemistry The enzyme galactosidase hydrolyzes lactose to yield galactose and glucose Analysis of this enzyme in E coli has shown that it is an inducible enzyme whose level of expression is dependent on substrate concentration The study of the lac operon has played an important role in under standing the control of gene expression in bacte ria In prokaryotes gene expression is controlled primarily at the level of transcription Ge
22. XP 2000 etc include HyperTerminal 0 Locate HyperTerminal hypertrm exe usually in Program files Accessories Run the program O The opening screen will request a name and icon for establishing a new connection Fig 4 In the Name field enter DQ 200 Near the left end of the generic icon set provided is a laboratory design Click on it Click OK to proceed to the next screen EEB 21x DyN quant Enter details for the phone number that you want to dial Country region United States of America 1 Phone number I Connect usina CT Cancel Fig 5 Select the appropriate COM port Area code el en a nern _ COMI Properties 22 1 Pott Settings Bits per second 1200 e Data bits a y Pat None Te Stopbits 1 Le Flow control a Fig 6 Connections and communication parameter setup Tools Table Window 1 Imre m Transfer Help Send File Receive File ture T Send Text File Capture to Printer Fig 7 Select Capture Text to record readings On connection screen Fig 5 select COM1 or the appropriate connection port The choices for telephone numbers will be inactivated and the small terminal screen will go blank Click OK to proceed to the next screen COM1 Properties Fig 6 Q Use the various drop down menus to set 1200 bits per second 8 data bits no parity 1 stop bit and no flow
23. ange of AT are available from Sigma Chemical Co See Appendix B for a partial listing H 33258 fluoresces only about half as much when it binds to single stranded genomic DNA as when it binds to double stranded genomic DNA Short pieces of single stranded DNA such as single stranded oligonucleotides however will not normally cause H 33258 to fluoresce in proportion to their concentration e Refer to DQ 200 Application Notes 8 and 11 listed in the ordering information section for more detailed information Factors with little or no effect e Buffers commonly used to extract DNA from whole cells e Low levels of detergent lt 0 01 SDS Generally the final detergent concentration should be well below the detergent s critical micelle concentration e High salt concentrations up to 3 M NaCl Note For full fluorescence a minimum amount of NaCl is required in the assay buffer At least 200 mM is required for purified DNA and 2 3 M is required for crude samples In crude samples higher salt concen trations appear to cause the dissociation of proteins from DNA making way for dye molecules RNA does not interfere significantly with the DNA assay because RNA does not generally bind H 33258 Under high salt conditions fluorescence due to RNA is usually well below 1 of that produced by the same concentration of DNA e p19 A Important Refer to the mate rial safety data sheet MSDS accompanying each chemi
24. aphing and linear regression analysis to determine the concentration Plot standard curve measurements to confirm linearity in the range of interest Important DQ 200 measurement notes e Accurate pipetting is critical Use a micropipetter accurate to 0 02 pl Turn the lamp on 15 minutes before use to allow the lamp and sample compartment temperature to stabilize Zero every blank assay solution before adding standard or sample Always orient the cuvette the same way Glass cuvettes usually have an identifying G on one side which can serve as an orientation guide If needed clean the sides of the cuvette with a low lint tissue Remove the cuvette from the well to add sample This reduces the risk of spilling solution into the well and reduces sample exposure to the lamp minimizing heating and photobleaching Always mix completely after adding standard or sample to the cuvette by drawing the solution into a disposable transfer pipet several times Do not introduce bubbles into the solution Always close the lid Repeat the measurement at each concentration at least once to verify that the results are reproducible Empty the cuvette between each measurement Rinse Drain the cuvette completely by blotting while inverted on a paper towel Fig 3 LCD screen display Numeric keys lt 0 gt lt 9 gt Use the numeric keypad to enter a calibration standard value or to choose menu options The lt ESC gt key
25. ard for fluorescent E coli genomic DNA measurements If you use 100 ng ml calf thymus DNA as a standard which has AT 58 but your sample is E coli DNA with 50 AT substitute into the equation Adjusted standard setting 2 5 AT sta AT samp 100 for 100 ng ml standard Adjusted standard setting 2 5 58 50 100 2 5 8 100 20 100 120 Set the DQ 200 Fluorometer to display 120 when measuring your 100 ng ml calf thymus DNA stan dard to read E coli DNA concentrations in ng ml Appendix C Main Bibliography Brunk C F et al Assay for nanogram quantities of DNA in cellular homogenates Anal Biochem 92 497 500 1979 Cesarone C Bolognesi C and Santi L Improved microfluorometric DNA determination in biological material using 33258 Hoechst Anal Biochem 100 188 197 1979 Daxhelet G A Coene M M Hoet P P and Cocito C G Spectrofluorometry of dyes with DNAs of different base composition and confor mation Anal Biochem 179 401 403 1989 Gallagher S In Current Protocols in Molecular Biology EA Ausubel et al A 3 9 A 3 15 Supplement 8 1989 Gallagher S ed GUS Protocols Using the GUS Gene as a Reporter of Gene Expression Academic Press Inc 1992 Jefferson R A Assaying chimeric genes in plants the GUS gene fusion system Plant Molecular Biology Reporter 5 387 405 1987 Labarca C and K Paigen A simple rapid and sensitive DNA assay procedure Ana
26. asure of confidence in the data If the measured values near one end of the range devi ate consistently from the best fit straight line the assay is being extended into a non linear region Samples should be diluted or assay conditions adjusted to return to a linear region of the plot A linear equation can be determined from a single reference point using O as the second point m y x b 0 however this will not give any indication that the assay might be out of the linear range 500 400 4 300 Fluorescence units 200 5 100 0 T T T T T 0 100 200 300 400 500 600 DNA concentration in the cuvette ng ml e p25 e p26 5 Enzyme Activity Quantitation Esters of 4 methylumbelliferone 4MU 7 hydroxy 4 methylcoumarin do not fluoresce unless cleaved to release the free fluorophore Free 4MU can be used as a standard to calibrate fluorometric enzyme assays based on the hydrolysis of 4MU containing substrates such as B 4 MU glucuronide by B glucuronidase GUS or B 4 MU galactose by B galactosidase A solution of 4MU can also be used to check instrument performance The accuracy of this assay is also dependent on the accuracy of the start and stop times for enzyme digestion of the substrate Specific protocols for assaying galactosidase and glucuronidase activities are in Appendix A Solutions 4MU stock solution A 1 mM 100 ml 4 methylumbelliferone sodium salt 19 8 mg MW
27. cal for detailed handling and safety information Hazard Hoechst 33258 is a possible mutagen Wear gloves and a mask and work under a fume hood e p20 Solutions Hoechst 33258 stock dye solution 1 mg ml Hoechst 33258 10 mg Distilled filtered water 10 ml Do not filter Store in an amber bottle at 4 C for up to 6 months 10X TNE buffer stock solution 100 mM Tris 10 mM EDTA 2 M NaCl 1000 ml Tris base Tris hydroxymethyl 12 11 g aminomethanel MW 121 14 EDTA disodium salt dihydrate 3 72 g MW 372 20 Sodium chloride MW 58 44 116 89 g Distilled water to 800 ml Concentrated HCI to pH 7 4 Distilled water to 1000 ml Filter before use 0 45 um Store at 4 C for up to 3 months Calf thymus DNA for the low range assay 1 10 dilution of standard stock 100 ug ml Calf thymus DNA standard 1 mg ml 100 ul 10X TNE 100 ul Distilled water filtered 800 ul Gently tap the tube to mix thoroughly Store at 4 C for up to 3 months Assay solution A for the low range DNA assay 10 500 ng ml final DNA conc 0 1 ug ml H 33258 in 1X TNE 0 2 M NaCl 10 mM Tris Cl 1 mM EDTA pH 7 4 H 33258 stock solution 10 ul 10X TNE 10 ml Distilled filtered water 90 ml Keep assay solution A at room temperature Prepare fresh daily Do not filter once dye has been added Assay solution B for the high range DNA assay 100 5000 ng ml final DNA conc 1 pg ml H 33258 in IX TNE 0 2 M NaCl 10 mM Tris Cl 1
28. dged from the bottom Turn the block over right side up to collect the mirror Handle with care Remove the reference and emission filter seal rings and place on a soft cloth The emission filter should slide out easily If required press the filter from behind with a cotton swab O Cleaning Clean the cuvette well with cotton swabs Dampen a soft cloth with alcohol and wipe each optical surface lf required gently polish with a dry soft cloth Remove all particles Allow to air dry All surfaces must be completely clean for accurate measurements e p31 A Reference mirror Fig 10 Optical block assembly e p32 Step 4 N excitation aperture I Y glass cover Step 3 Mn L f Optical block _ 1 bottom view Sample mirror Step 5 Optical block Cuvette well top view Seal ring Reference mirror Emission filter 9 Arrow points away gt from optical block Seal ring Emission filter E Thumb screw Phillips screw NW Ground plate Optical block assembly If the reference mirror was removed choose the best surface to face the reference beam Slide the mirror into the slot until it stops The mirror self aligns 2 Hold the block in your palm as in step 3 above slide the glass cover into the excitation aperture slot and seat the ground plate onto the optical block Secure with the Phillips screw Carefully slide the sample mirror into the cuvette well The
29. e actual concentration of the standard Low range assay calibration value High range assay calibration value 100 ng ml 1000 ng ml Or enter a convenient value that will display a multiple of the actual DNA concentration Press lt ENTER gt Calibration Tip The suggested calibration procedure sets the instrument to display the DNA concentration of the solution in the cuvette in units of ng ml This corre sponds to a concentration of ug ml of DNA from the sample tube if 2 ul of sample is used This is a 1 1000 dilution of sample into assay solution This relationship only holds if the volumes for both the standard and the unknown DNA sample are the same That is if you set the instrument with a different volume of standard use the new volume for the DNA sample also to preserve the relationship Ps E Measure the fluorescence of the unknown sample Remove the cuvette drain and rinse Add 2 ml assay solution and place the cuvette into the well Zero the instrument as in step 1 Remove cuvette add 2 ul of the unknown DNA sample mix thoroughly by pipetting with a disposable transfer pipette do not introduce bubbles Place the cuvette in the well and close the lid to display sample fluorescence Dots flash in the lower left corner until the measure ment stabilizes Record the value displayed Drain the cuvette lf desired repeat the measurement with a second sample and average the readings If the unknown sample
30. e stop buffer 1 9 ml GUS extraction buffer 50 mM NaHPO4 pH 7 0 10 mM 2 mercaptoethanol 10 mM NazEDTA 0 1 sodium lauryl sarcosine 0 1 Triton X 100 For 100 ml extraction buffer mix 1M NaHPOa pH 7 0 5 00 ml 2 mercaptoethanol 0 07 ml 0 5 M Na2EDTA pH 8 0 2 00 ml 30 Sarcosyl 0 33 ml 10 Triton X 100 1 00 ml Distilled water 91 60 ml GUS assay buffer 2 mM MUG in extraction buffer To prepare 25 ml assay solution mix 4 methylumbelliferyl D glucuronide 25 mg Extraction buffer 25 ml Note The water content of MUG preparations may vary For greatest accuracy the calculation of solution molarity should take this into account e p39 e p40 Generate a concentration curve Follow the instructions in section 5 to calibrate the instrument and generate a concentration curve using the 4 MU standard This procedure demon strates the linearity of readings in the expected range of the assay Time course assay The routine MUG assay is based on a linear rate of substrate hydrolysis as a function of time The control assay listed below uses a commercial suspension of GUS diluted into extraction buffer to demonstrate linearity The linearity of the assay over time is critical and should be verified under your specific conditions Once you have demonstrated that the system is linear in time substitute aliquots of unknown sample extracts for the diluted commercial enzyme to determine the level of GUS expressio
31. e value Zero using blank capillary assay solution and recalibrate The entered calibration value is too high Recalibrate with standard solution Use a lower factor if not using the actual standard concentration The diagnostic test failed Call the Hoefer Inc Technical Service Department MUG non fluorescent DI Appendix A Enzyme assay protocols glucuronidase Assay B Glucuronidase GUS is the reporter enzyme of choice for much plant genetic research The E coli gene for GUS was originally isolated by R A Jeffer son and co workers Jefferson 1989 and is now commercially available from Clontech Laborato ries in a variety of configurations The recombinant GUS gene mRNA and enzyme can be routinely manipulated and assayed to study promoter func tion tissue specific expression developmental regulation mRNA stability excision events of transposable elements and signal sequences that target proteins for various organelles The advantages of using GUS to report the activ ity of various promoters and genes are two fold first with few exceptions plants lack GUS activ ity and second GUS assays are straightforward with substrates suitable for both histochemical and enzymatic analysis readily available from a vari ety of companies including Molecular Probes Research Organics and Clontech Typically GUS activity in solution is determined with the fluoro genic substrate 4 methylumbelliferyl B D g
32. efer Inc si riserva il diritto di apportare modifiche ai dati tecnici senza preavviso Garanzia e responsabilit Hoefer Inc garantisce che prima della consegna il prodotto stato collaudato a fondo per soddisfare i requi siti specificati La garanzia inclusa nelle condizioni di consegna risulta valida solamente se il prodotto stato installato ed utilizzato nel rispetto delle istruzioni fornite da Hoefer Inc Hoefer Inc non potr essere ritenuta responsabile di incidenti o danni consequenziali inclusi ma non limitati a perdite di profitti mancato guadagno perdite di affari difetti di funzionamento e relative esposizioni dovuti ad un utilizzo non corretto del prodotto 2004 Hoefer Inc Tutti i diritti riservati Nessuna parte della presente pubblicazione pu essere riprodotta conservata in sistemi di gestione dati o trasmessa in alcun forma n per nessuno scopo senza autorizzazione scritta del produttore Note Fluorescent emission output is not strictly linear and it is affected by numerous variables If the procedures in this manual are followed closely accurate concentration measurements can be made with a high degree of reliability 1 Fluorometer function and description The Hoefer DQ 200 Fluorometer is a filter fluorescence photometer with a fixed excitation bandpass source 365 nm and an emission band pass filter 460 nm It is designed specifically for the accurate quantitation of l
33. emove the cuvette and add the corresponding volume of standard Mix thor oughly and place the cuvette into the well in its original orientation close the lid and record the display value Measure a second sample for each value sample 2 and average the two readings Carbonate 1uM 4MU Sample conc Reading Reading Avg reading buffer stock B xX sample 1 sample 2 y vol ml vol ul nM samples 1 2 2 2 0 0 0 1 9 100 50 1 8 200 100 1 7 300 150 1 6 400 200 a e p28 IE Plot the 4MU concentration x vs the averaged readings y The resulting graph should be linear as shown in Fig 9 See page 25 for methods to analyze the data E 2000 Fig 9 4 MU standard curve Fluorescence increases linearly with concentration 1500 gt a 2 D E 8 1000 2 8 8 S E mi 500 0 0 50 100 150 200 Methylumbelliferone nM e p29 A Important e Turn the mains power off and unplug the power cord e The optical surfaces are easily scratched Handle with extreme care and polish gently e Wear gloves when servic ing the optical block This protects both the technician from hazardous materials that may have been spilled and protects the optical surfaces from fingerprints e Use only isopropanol on a clean soft cloth to clean optical surfaces e p30 6 Care and maintenance Cleaning To clean the exterior wipe the unit with a damp cloth Never use abrasive cleansers or
34. instalado y utilizado de acuerdo con las instrucciones entregadas por Hoefer Inc Hoefer Inc no ser responsable bajo ning n concepto de da os directos o indirectos incluyendo sin limit aci n la p rdida de beneficios la p rdida de ingresos la p rdida de oportunidades de negocio la p rdida de utilizaci n y otras consecuencias relacionadas cualqui era que sea la causa que se deban a la utilizaci n defectuosa e incorrecta del producto O 2004 Hoefer Inc Reservados todos los derechos No est permitida la reproducci n ni el almacenaje en un sistema de recuperaci n ni la transmisi n de parte alguna de esta publicaci n sin la autorizaci n por escrito de la empresa Deutsch Wichtige benutzerinformationen F r ein vollst ndiges Verst ndnis und eine sichere Handhabung dieses Produktes ist es notwendig da der Benutzer dieses Handbuch vollst ndig durchliest Ein Ausrufezeichen in einem gleichseitigen Dreieck soll den Benutzer auf die Anwesenheit wichtiger Betriebs und Wartungsanweisungen in der dem Ger t beiliegen den Dokumentation hinweisen Wenn Sie Anmerkungen zu diesem Handbuch haben dann senden Sie diese bitte an Hoefer Inc 953 Indiana Street San Francisco CA 94107 USA support hoeferinc com Hoefer Inc beh lt sich das Recht vor die Spezifika tionen ohne vorhergehende Ank ndigung zu ndern Gew hrleistung and haftung Hoefer Inc garantiert da das geliefer
35. ion Close the lid Press lt ENTER gt Displays fluorescence The assay solution background is determined and subtracted Press lt ZERO gt Remove the cuvette Add sample to the cuvette and mix Do not introduce bubbles Place the cuvette back in the well in the same orientation Close the lid and press lt ENTER gt Displays fluorescence in chosen units Press lt ENTER gt Choose lt 1 gt to measure the next sample Choose lt 2 gt to calibrate to a different standard Choose lt ESC gt to return to the Main Menu 1 gt Prompt 2 gt Units 3 gt Send 4 gt More Prompt 1 gt 0ff 2 gt 0n Units 1 gt ng ml 2 gt None 1 gt Manual send 2 gt Auto send Main menu option 2 gt Setup Select lt 2 gt from the Main Menu to access the Setup menu which accepts operator preferences Each submenu is described below Press lt ESC gt at any time to return to the Main Menu Press the number associated with the parameter of interest to access the following submenus 1 gt Prompt of f displays only measurements and minimal instructions 2 gt Prompt on guides the user through the assay step by step 1 gt Sets units to display ng ml 2 gt No units will be displayed After a brief pause the Setup menu is displayed 1 gt The Manual send option sets the software so that measurements and the corresponding ID numbers are transmitted to the serial port only
36. l Biochem 102 344 352 1980 Marmur J and Doty P Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature J Molec Biol 5 109 118 1962 Moe D Garbarsh C and Kirkeby S The Protein Effect on Determination of DNA with Hoechst 33258 J Biochem Biophys Methods 28 263 276 1994 Rymaszewski A et al Estimation of cellular DNA content in cell lysates suitable for RNA isolation Anal Biochem 188 91 96 1990 Stout D L and EF Becker Fluorometric quantita tion of single stranded DNA a method appli cable to the technique of alkaline elution Anal Biochem 127 302 307 1982 e pol Ordering information product quantity code number Basic Unit Hoefer DQ 200 Fluorometer 1 DQ200 Includes DNA standard and Hoechst 33258 dye 100 mg 115 230 VAC Glass fluorometry cuvette fluorescent grade 1 DQ105 Lamp replacement assembly 1 DQ200RK 3 Optics replacement kit Includes filter 1 DQ200RK 5 glass cover mirrors and O ring Lid replacement assembly Includes lid 1 DQ200RK 4 latch spring and mounting screw Capillary Adaptor Capillary Adaptor Kit 1 DQ120 Includes capillary tubes 10 50 and 100 ul 20 each Capillary tubes 10 pl 100 DQ120 10 Capillary tubes 50 pl 100 DQ120 50 Capillary tubes 100 pl 100 DQ120 100 Capillary Cuvette Capillary Cuvette Adaptor Kit 1 DQ130 Includes one capillary cuvette capillary tubes 9 ul pkg of 25
37. l sample should contain 10 to 10 units nM 4MU min of B galactosidase activity If other dilutions of the sample into the reaction cocktail are used adjust samples accordingly O The glycine carbonate reagent is sufficient to titrate 1 3 its own volume in 5 TCA to proper pH for reading 200 ul reaction stopped with 50 ul of 25 TCA The protocol is designed for assay of E coli fB galactosi dase activity which is active at neutral pH The vertebrate form of f galactosidase is a lysosomal enzyme which has optimal activity at pH 4 5 The lysosomal activity can be assayed in acetate buffer instead of Tris buffer The assay should be performed at both pH values when lysosomal contamination of reactions is anticipated O Depending on the calibration setting used maximum readings are obtained at 40 to 200 nM 4MU final concentration in the glycine carbonate buffer Limitations on the sensitivity of the assay are deter mined by the background fluorescence of the substrate It is therefore important to use freshly prepared substrate solutions in assays where high sensitivity is desired O Frozen reaction cocktails may be used but backgrounds gradually increase with repeated freeze thaw cycles Substrate sometimes precipitates from frozen cocktails Resolubilization is slow unless the cocktail is heated to 37 C and repeatedly vortexed Bibliography An G Hidaka K and Siminovitch L Exp
38. low its critical micelle concentra tion Final Triton X 100 conc must be below 0 001 Use a standard with a G C content very similar to that of your sample Single stranded genomic DNA yields about half the fluorescence of an equal amount of double stranded DNA Therefore ssDNA standard should be used for ssDNA samples If the sample contains a very high protein concentration which may produce high background fluorescence pretreating the sample with 0 1 0 5 mg ml proteinase K in pronase and adding 2 3 m NaCl to the assay solution has been reported to lower the background Moe et al 1994 e p35 Error and other messages One dot remains in the left corner Turning lamp on Zero first using a blank sample Blank gt sample Zero and re calib Re calib using lower value Failed e p36 A series of dots across the display indicate that the instrument is stabilizing a measurement If one dot remains in the left corner no stable reading was determined Wipe the cuvette and repeat the measurement procedure The lamp was inadvertently turned off or the auto shut function switched the lamp off after one hour of no keypad activity This message reports that the lamp is being turned on Wait 15 minutes before taking measurements to allow the lamp to stabilize Assay solution was not blanked Zero the instrument place blank assay solution into cuvette and press lt ZERO gt Blank value is higher than the sampl
39. lucuro nide MUG glucuronic acid 4MU fluorescent The reaction product 4 methylumbelliferone 4MU is maximally fluorescent at high pH where the hydroxyl group is ionized Addition of a basic solution of sodium carbonate simultaneously stops the assay and adjusts the pH for quantitat ing the fluorescent product The Km for the MUG substrate in this assay is 0 6 to 0 7 mM with a minimum detectable GUS activity of 2 pmol MUG hydrolyzed E coli GUS has a molecular mass of 68 2 kDa and under some conditions of SDS PAGE an apparent molecular mass of 74 kDa e p37 EF Materials Enzyme control B glucuronidase GUS liquid suspension from E coli Roche Substrate 4 methylumbelliferyl B D gluc uronide MW 352 3 Clontech 8082 Molecular Probes M 1490 Roche Sigma Chemical Co M 9130 Calibration standard 4 methylumbelliferone sodium salt MW 198 2 4MU 7 hydroxy 4 methylcoumarin methylumbelliferone Reagents Na2HPO4 MW 141 96 NaH2P0O4 MW 119 98 Na2C03 MW 105 99 Na2EDTA MW 372 24 Sarcosyl 2 mercaptoethanol Triton X 100 Equipment DQ 200 Fluorometer glass cuvette If using disposable plastic cuvettes order fluores cence grade e p38 Solutions 4 Methylumbelliferone standard 50 nM 4MU in carbonate stop buffer Prepare standard stock solutions and carbonate stop buffer as described on page 26 Prepare 50 nM 4MU standard solution just before use 1 uM 4MU solution 100 ul Carbonat
40. ml Reaction cocktail 20 ml Prepare the reaction cocktail minus substrate at room temperature The substrate 4 MUG is most easily dissolved in cocktail by first dispersing in absolute ethanol 1 M Tris HCI pH 7 5 25 mM 0 5m 5 M NaCl 125 mM 0 5m 0 1 M MgClo 2mM 0 4 m 2 mercaptoethanol 12 mM 17 0 u Distilled water 18 5 m 4 MUG FW 338 3 0 3 mM 100 y methylumbelliferyl B D galactoside Dissolve 2 mg into 100 ul abs EtOH 0 5 of the total volume of cocktail and then rapidly vortex into the aqueous cocktail solution until dissolved TCA solution 25 w v trichloroacetic acid Generate a concentration curve Follow the instructions in section 5 to calibrate the instrument and generate a concentration curve using the 4 MU standard This procedure demon strates the linearity of readings in the expected range of the assay Assay 0 Add 40 ul of sample to a microcentrifuge tube Use H20 for a blank 9 Add 160 ul of the reaction cocktail Incubate at 37 C for 30 minutes 4 Stop the reaction by adding 50 ul of 25 TCA Cool on ice Clarify the solution by centrifugation in a microcentri fuge for 1 to 2 minutes O Add 0 1 ml of supernatant to 1 9 ml of glycine carbonate reagent O Read the fluorescence Determine the concentration from the standard concentration curve of O to 200 nM 4MU p45 e p46 Notes on the standard procedure 0 A 40 u
41. n in your experimental system Depending on the level of GUS gene expression you may need to dilute samples further or to allow the reaction to proceed longer to generate results in the linear range of the assay If high levels of activity precision are not required single rather than multiple time points may be used Enzyme activity units are normally expressed in nmol product released per minute per pg of protein This value is the slope of the line plotted through the time points divided by the amount of protein added Dilute 10 ul of the commercial enzyme stock into 1 ml extraction buffer for a 1 100 dilution Further dilute this by adding 10 ul of the 1 100 dilution to 10 ml of extraction buffer to achieve a 1 100 000 dilution Keep enzyme stocks on ice Use this final dilution for subsequent assays DP Y To two test tubes on ice add Assay solution 250 ul Extraction buffer 200 ul GUS enzyme diluted 1 100 000 50 ul For a final concentration of 1 mM MUG Remove duplicate 50 pl aliquots for the reagent blank These should be quenched immediately in 2 ml carbonate stop buffer 4 Remove the test tubes containing the assay solution from the ice and start the assay by placing them in a 37 C water bath Stagger successive tube assays by 30 second intervals For each time point transfer 50 pl aliquots from each test tube staggered by 30 seconds into 2 ml stop buffer to quench the reaction
42. ndard assay and sample solu tions See Section 4 for DNA quantitation and Section 5 for GUS assays 2 To change settings to different operator preferences Choose 2 gt Setup to select options such as prompt mode concentration units and auto send To calibrate the instrument Press lt 1 gt Read Set the zero by inserting the blank cuvette containing the assay solution without standard or sample Close the lid Press lt ZERO gt After O is displayed remove the cuvette add the proper standard to the cuvette and mix Insert cuvette into the well and press lt CALIB gt Enter the standard concentration and press lt ENTER gt 0 To measure the fluorescence of the sample Set the zero by inserting the blank cuvette containing assay solution without sample Press lt ZERO gt After O is displayed remove the cuvette add the sample solu tion and mix well Insert the cuvette into the well close the lid and record the measurement Closing the lid is equivalent to pressing lt ENTER gt The sample can be read again by pressing lt ENTER gt Continues on following page St O Analyze the results If following the protocols as described the actual concen tration is displayed automatically If deviating from the protocols by using different concentrations or a calibration value that is a factor of the standard concentration use mathematical tools such as gr
43. neticists have developed a variety of colored indicator assays that indicate the level of B galacto sidase expression These methods may provide extra qualitative or quantitative information as a chromo genic lactose analog is cleaved by galactosidase The promoter activity of mammalian genes can be analyzed by using fusion genes containing the promoter of interest attached to the bacterial B galactosidase gene The level of B galactosidase expression indicates the level of transcription under different regulatory conditions Sensitive and quantitative assays of B galactosidase activity are often needed The following assay measures the hydrolysis of the fluorogenic B galac tosidase substrate Cleavage of 4 methylumbel liferyl B D galactoside by galactosidase yields the fluorescent molecule 4 methylumbelliferone 7 hydroxy 4 methylcoumarin 4MU The 4 methylumbelliferone moiety is fluorescent above pH 8 When excited by 365 nm light 4MU emits light at 460 nm The assay is sufficiently sensitive to detect picogram quantities of B galactosidase e p43 e p44 Solutions Glycine carbonate stop buffer 1 liter Glycine 133 mM 75 1 g Na2CO3 83 mM 106 0 g pH to 10 7 4 Methylumbelliferone standard 50 nM 4MU in glycine carbonate stop buffer 2 ml Prepare 4MU stock solutions as described on page 26 Prepare 50 nM 4MU standard solution just before use 1 uM 4MU solution 100 0 ul Glycine carbonate stop buffer 1 9
44. nt the reading will first drift upward as light is scattered by the bubbles until they move out of the beam range or dissipate If particulates are present the reading may suddenly rise as a particulate drifts in the light path and then drop as it moves out of the beam range Thoroughly mix the sample and assay solution by gently pipetting into a disposable transfer several times without intro ducing bubbles Use a micropipet accurate to 0 02 ul If inconsistencies persist either use larger aliquots or dilute the sample in the appropriate buffer Use a larger sample Use only pure distilled and filtered 0 2 or 0 4 um filter water or all solutions Filter the 1X TNE working buffer to remove all particulates Particulates may cause light to scatter causing measurement fluctuations Filter the buffer before adding H 33258 because the dye binds to most membrane types Wipe the outside of the cuvette before placing it into the sample chamber problem solution Readings negative or lower than expected Crude cell lysates prepared with acid guanidinium thiocyanate phenol solution Use the appropriate reference standard Readings higher than expected Use freshly prepared assay solution at ambient temperature to set the zero and for all subsequent measurements Extract ethidium bromide from DNA solutions because ethidium bromide interferes with the fluorescence of H 33258 H 33258 is useful only for
45. o that the back vents are not obstructed Use and store the instrument away from direct sunlight and away from areas where the instrument may become wet Allow 15 minutes for the lamp to warm up each time it is switched on Do not add more than 2 ml of solution to the cuvette Wipe the cuvette exterior before placing it into the well Take care not to spill any liquid into the well Reliable results depend on measurement accuracy and consistency For the DNA assay use a pipette accurate to 0 02 ul and always use the same amount of assay solution For instance if using the glass cuvette always add 2 ml The optical surfaces must remain clean in order to measure fluorescence accurately Periodically clean the optical surfaces as described in the care and maintenance section If this equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired Only accessories and parts approved or supplied by Hoefer Inc may be used for operating maintaining and servicing this product Instrument set up Mains power Plug one end of the power cord into the receptacle on the back of the unit marked MAINS Plug the other end to a suitable grounded power outlet 2 Turn the mains power switch beside the power cord receptacle to on I See section 3 for complete operating instructions Serial port connector The RS232C serial port is a DB9 9 pin male
46. or to calibrate the instrument Select off when using a capillary cuvette or capillary adaptor because the instrument is not zeroed in the same manner as when using the 2 ml cuvette LCD message Concentration Computing zero Concentration Enter standard conc Calibrating Concentration Concentration Action required Displays fluorescence in chosen units To zero the instrument Add only assay solution no standard to the cuvette and place it into the cuvette well always in the same orientation Close the lid and press lt ZERO gt Displays fluorescence in chosen units To calibrate the instrument Add the appropriate standard amount and concentration to the cuvette Mix by drawing the solution into a disposable transfer pipet several times Do not introduce bubbles into the solution Place cuvette in well always in the same orientation close the lid and press lt CALIB gt Enter the concentration of the stan dard Press lt ENTER gt Displays fluorescence in the chosen units To measure fluorescence Remove the cuvette rinse and drain Add only assay solution no sample to cuvette Place cuvette in well always in the same orientation and zero the instru ment as before Remove cuvette add sample and mix Place cuvette in well close lid and record the measurement Displays fluorescence in chosen units Press lt ENTER gt to re read the sample concentration
47. ount added 2 ul x 100 ng ul 200 ng final concentration 200 ng 2 ml 100 ng ml Fig 8 Concentration curve using calf thymus DNA for low range assay Data analysis example Duplicate data points were plotted and analyzed by a linear least squares regression Best fit parameters r 0 9998 b 0 95 m 1 00 y 1 00x 0 95 To find an unknown DNA concentration assign the display value to the y variable and solve for the DNA concentration x Analyze the results 0 Plot the sample concentration x vs the averaged reading y Your data may be slightly lower or higher than expected but as long as the plot is linear you can expect accurate values for unknown samples within the range of the standard curve Slight variations are most commonly due to pipetting variability The accuracy of this assay also depends on the accuracy of the start and stop times for the digestion O Determine the equation of the straight line Either draw a straight line through the data with an estimated best fit or calculate a least squares best fit for the data A linear regression is quickly accomplished with any math program The line is described by the equation y mx b where y is the instrument reading x is the known DNA concentration m is the slope of the line and b is the y axis intercept For ideal data m 1 and b O Statis tical analysis of the error in the fit gives a correlation coefficient r a me
48. ow DNA concentra tions using Hoechst 33258 dye The instrument can also measure enzyme activity based on the cleavage of coumarin methylumbelliferyl linked substrates as well as other fluorescent assays for which these excitation and emission wavelengths are appropriate A calf thymus DNA standard and the Hoechst 33258 dye are included to provide a reference point to calibrate the instrument The sample measurement cell cuvette capillary adaptor or capillary cuvette is ordered separately A glass cuvette is recommended for 2 ml assays Two kits for handling micro samples are available The Capillary Adaptor Kit includes a capillary adap tor and capillary tubes for 10 to 100 pl of solu tion The Capillary Cuvette Adaptor Kit includes a focusing cuvette cell holder which provides increased sensitivity and capillary tubes for 3 to 9 ul of solution Unpacking Unwrap all packages carefully and compare contents with the packing list making sure all items arrived If any part is missing contact your local sales office Inspect all components for damage that may have occurred while the unit was in transit If any part appears damaged contact the carrier immediately Be sure to keep all pack ing material for damage claims or for repacking should it become necessary to return the unit Fig 1 Main components The power switch power cord receptacle and communications port are on the rear panel LCD screen Included b
49. res sion of Bacterial galactosidase in Animal Cells Mol Cell Biol 2 1628 1632 1982 Beckwith J and Zipser D eds The Lactose Operon Cold Spring Harbor Laboratory Cold Spring Harbor New York 1970 Miller J Experiments in Molecular Genetics Cold Spring Harbor Laboratory Cold Spring Harbor New York 1972 Miller J The Operon Cold Spring Harbor Labo ratory Cold Spring Harbor New York 1981 Ullman A Jacob E and Monod J Charac terization by in vitro complementation of a peptide corresponding to an operator proximal segment of the galactosidase structural gene of Escherichia coli J Mol Biol 24 339 343 1967 e p47 e p48 Appendix B Effect of A T content on fluorescent DNA quantitation Dealing with A T content differences when using the H 33258 DNA assay Because fluorescence enhancement with H 33258 occurs only when the dye is bound to A and T bases of a double stranded DNA chain the intensity of the fluorescent signal is determined by both the concentration of the DNA and the A T content AT of the DNA The H 33258 fluorescence assay must be calibrated with a DNA standard of known concentration which has been determined by UV absorbance If the AT of the standard and the sample are similar no correc tion for the base composition is required So when measuring eukaryotic DNA which has an AT ranging from 56 to 60 the standard DNA is typically calf thymus with an AT
50. s Finally a 20 second countdown appears as the system warms up The Main Menu appears when the instrument is ready to receive input Main menu The Main Menu accesses three different functions 1 gt Read measures fluorescence 2 gt Setup sets operator preferences and 3 gt Test runs a comprehensive diagnostic routine Press lt ESC gt anytime to return to this screen The following three sections describe all options in the Main Menu You may wish to set opera tor preferences see the section titled Main menu option 2 gt Setup before working through the 1 gt Read section which prepares the instru ment to measure fluorescence Main menu option 1 gt Read Press lt 1 gt to select the read option that prepares the instrument to measure fluorescence This can be performed with either Prompt o f f which is the default setting and does not guide the operator at each step or the Prompt on which displays each step of the DNA assay Select from the 2 gt Setup menu The display for each option is fully described in the following two pages Once you are familiar with the instrument you will probably choose prompt of f for routine measurements Note Pressing the enter key at every step is not required as it is in Prompt mode on Prompt off Prompt of f is the default setting In this mode the operator is not prompted to zero every assay solution
51. solvents The only user serviceable component is the optical block Optical block assembly is described in the cleaning section below Optical block Clean the optical block periodically depending on the frequency of use or if solution spills into the cuvette well Optical block disassembly 0 Turn the mains power off and unplug the power cord Spread a soft cloth over the work area and turn the unit upside down onto the padded surface Wear gloves both to protect yourself and the optical surfaces O Locate the thumb screw near the front of the unit and unscrew The captive thumbscrew stays attached to the block Lift the optical block assembly straight up Hold the optical block assembly so that the ground plate with the thumbscrew faces up and the opti cal block is cradled in your palm In this position no components will be damaged if they slide out of their slots during disassembly Unscrew the Phillips screw near the thumbscrew Lift the ground plate and remove the glass cover in front of the excitation aperture Keep the optical block in this position for steps 4 and 5 SO The stainless steel reference mirror does not contact solution so it requires little maintenance If it requires cleaning insert a hook such as a paper clip in the hole where the mirror bends and pull the mirror out The sample mirror which covers two sides of the cuvette well slides out when gently nu
52. te Produkt sorg f ltig auf die Einhaltung der ver ffentlichten Spezifi kationen getestet wurde Die in den Lieferbedingungen n her erl uterten Gew hrleistungsanspr che gelten nur dann wenn das Produkt gem den von Hoefer Inc gelieferten Anweisungen installiert und benutzt wurde Hoefer Inc bernimmt keinerlei Haftung f r Sch den oder Folgesch den einschlie lich aber nicht begrenzt auf Gewinneinbu en Einkommensverluste entgangene Gesch ftsabschl sse Verlust der Gebrauchsf hig keit oder andere Verluste die wie auch immer durch eine fehlerhafte oder unsachgem e Verwendung des Produkts verursacht wurden 2004 Hoefer Inc Alle Rechte vorbehalten Die vorliegende Ver ffentlichung darf nur mit vorhergehender schriftlicher Genehmigung durch das Unternehmen vervielf ltigt in einem Abrufsys tem gespeichert oder in irgendeiner Form oder mit irgendwelchen Mitteln bertragen werden Italiano O pvi A Informazioni importanti per l operatore Per un utilizzo sicuro del prodotto leggere attentamente l intero contenuto del presente manuale II punto esclamativo all interno di un triangolo equilatero indica all operatore la presenza di importanti istruzioni di funzionamento e manutenzione nella documentazione allegata al prodotto Si prega di inviare eventuali commenti al presente manuale a Hoefer Inc 953 Indiana Street San Francisco CA 94107 USA support hoeferinc com Ho
53. toute perte de perspec tives commerciales tout emp chement d utilisation et tout autre risques ayant un rapport avec l utilisation du produit mais sans aucune limitation quant la nature de ces dommages O 2004 Hoefer Inc Tous droits r serv s La reproduction le stockage dans un syst me de r cu p ration d informations ou la transmission sous quelque forme que ce soit et par quelque moyen que ce soit de la pr sente publication en totalit ou en partie sont strictement interdits sans autorisation pr alable crite de la soci t piii Espanol O piv A Informaci n importante para el usuario Para comprender el producto y utilizarlo con seguridad es necesario leer este manual en su totalidad El signo de admiraci n en un tri ngulo equil tero en el manual advierte al usuario sobre la presencia de instrucciones importantes de operaci n y mantenimiento del aparato Si desearan hacer alg n comentario sobre este manual tengan la amabilidad de remitirlo a Hoefer Inc 953 Indiana Street San Francisco CA 94107 USA support hoeferinc com Hoefer Inc se reserva el derecho a modificar las espe cificaciones sin previo aviso Garantia y responsabilidad Hoefer Inc garantiza que el producto entregado ha sido probado a fondo para comprobar el cumplimiento de las especificaciones publicadas La garantia incluida en las condiciones de entrega s lo es v lida si el producto se ha
54. uctions accompanying the standard precisely to achieve the proper dilution e p21 A Important Accurate pipetting and thorough mixing are criti cal for reproducible results use a micropipetter accurate to 0 02 pl Orient the cuvette the same way each time you place it in the sample chamber Glass cuvettes usually have an identifying G on one side which can serve as an orientation guide The fluorescence measurement stabilizes quickly and then begins to drop as the sample warms in the chamber A series of dots across the display indicate that the instru ment is stabilizing a measure ment If one dot remains in the left corner check the Trouble shooting section e p22 The following steps assume the prompt is off If unfamiliar with the instrument you may wish to turn the prompt mode on by choosing 2 gt Setup from the Main Menu and then pressing 1 gt Prompt then 2 gt 0n 0 Zero the instrument with the assay solution Add 2 ml of the proper assay solution to the cuvette insert the cuvette into the well always in the same orientation close the lid and press lt ZERO gt After 0 appears remove the cuvette 2 Calibrate the unit Add 2 ul of low or high range standard solution to 2 ml assay solution in the cuvette mix by pipetting into a disposable transfer pipette several times place cuvette in well close the lid and press lt CALIB gt Enter th
55. ut not shown e Calf thymus DNA standard e Hoechst 33258 dye Required but not included Fluorometry cell left to right glass cuvette capillary adaptor or capillary cuvette The cell fits into the cuvette well under the lid Access the cuvette well by pressing the lid release This declaration of conformity is only valid for the instrument when it is e used in laboratory locations and e used as delivered from Hoefer Inc except for alterations described in the user manual Specifications Power input rating Fuse value Light source Lamp output Emission filter Environmental operating conditions Dimensions h x w x d Product certifications 115 V or 230 V 47 63 Hz T 3 154 250V microfuse Mercury lamp expected life 5000 hr 365 nm 7 nm 460 nm 15 nm Indoor use 15 40 C dry area away from intense light such as direct sunlight Humidity less than 80 for 5 31 C decreasing linearly to 50 for 31 40 C Altitude up to 2000 m Installation category Il Pollution degree 2 13 x 16 5 x 35 cm UL 61010A 1 CSA C22 2 1010 1 CE Certified Important information e Hoechst 33258 dye is a possible mutagen Wear gloves when handling Wear a mask when weighing Disposal must comply with all applicable regulations Never dispose of by pouring into a drain Always unplug the instrument before removing the bottom panel or cleaning the instrument Place the instrument s
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