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1 Fluorometer Function and Description Unpacking

1. A 5 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 RA 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 p Galactosidase Assay Contributed by William A Braell Harvard Medical School Department of Biological Chemistry The enzyme P galactosidase hydrolyzes lactose to yield galactose and glu cose Analysis of this enzyme in E coli has shown that it is an inducible enzyme whose level of expression is dependent on substrate concentra tion The study of the lac operon has played an important role in under standing the control of gene expression in bacteria In prokaryotes gene expression is controlled primarily at the level of transcription Geneticists have developed a variety of colored indicator assays that indi cate the level of B galactosidase expression These methods may provide extra qualitative or quantitative information as a chromogenic lactose analog is cleaved by B galactosidase The promoter activity of mammali
2. ment interdits sans autorisation pr alable crite de la soci t Gew hrleistung and Haftung Amersham Biosciences garantiert da das gelieferte Produkt sorgfaltig auf die Einhaltung der ver ffentlichten Spezifikationen getestet wurde Die in den Lieferbedingungen n her erl uterten Gewahrleistungsanspriiche gelten nur dann wenn das Produkt gem den von Amersham Biosciences gelieferten Anweisungen installiert und benutzt wurde Amersham Biosciences bernimmt keiner lei Haftung f r Sch den oder Folgesch den einschlie lich aber nicht begrenzt auf Gewinneinbufien Einkommensverluste entgan gene Gesch ftsabschl sse Verlust der Gebrauchsfahig keit oder andere Verluste die wie auch immer durch eine fehlerhafte oder unsachgem e Verwendung des Produkts verur sacht wurden Amersham Biosciences 1998 Alle Rechte vorbehalten Die vorliegende Ver ffentlichung darf nur mit vorhergehender schriftlicher Genehmigung durch das Unternehmen vervielf ltigt in einem Abrufsystem gespeichert oder in irgendeiner Form oder mit irgendwelchen Mitteln bertragen werden English Important user information Please read this entire manual to fully under stand 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 mainte nance instructions in the literature accompanying the instrument Should yo
3. 7 1 Wide fluctuations in fluorescence values Y V Thoroughly mix the sample and assay solution by gently pipetting into a disposable transfer several times without introducing bubbles Use a micropipet accurate to 0 02 ul If inconsistencies persist either use larger aliquots or dilute the sample in the appropri ate buffer Use a larger sample Use only pure distilled and filtered 0 2 or 0 4 um filter water for 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 Readings negative or lower than expected 7 2 V V V Use freshly prepared assay solution at ambient temperature to set the zero and for all subsequent measurements Extract ethidium bromide from DNA solutions because ethidi um bromide interferes with the fluorescence of H 33258 H 33258 is useful only for measuring DNA concentrations Spectrophotometer measurements of A260 and Auen 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 fluorometric value is usually more accurate provid ed a clean DNA standard of known concentration is used Crude cell lysates
4. 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 F F Becker Fluorometric quantitation of single stranded DNA a method applicable to the technique of alkaline elution Anal Biochem 127 302 307 1982 C 1 Customer Service Information Technical Service and Repair Amersham Biosciences offers complete technical support for all our products If you have any questions about how to use this product or would like to arrange to repair it please call or fax your local Amersham Biosciences representative Important Request a copy of the Amersham Biosciences Health and Safety Declaration Form before returning the item No items can be accepted for servicing or return unless this form is properly completed Ordering Information Basic Unit Hoefer DyNA Quant 200 Fluorometer Includes DNA standard and Hoechst 33258 dye 100 mg 115 230 V 1 80 6406 80 Glass fluorometry cuvette fluorescent grade 1 80 6227 44 Lamp replacement assembly 1 80 6228 96 Optics replacement kit Includes filter glass cover mirrors and O ring 1 80 6229 34 Lid replacement assembly Includes lid latch spring and mounting screw 1 80 6229 53 Capillary Adaptor Capillary Adaptor Kit 1 80 6227 63 includes capillary tubes 10 50 and 100 ul 20 each Capillary tubes 10 ul 100 80 6227 82 Capillary tubes 50 ul 100 80 6228 20
5. Anmerkungen zu diesem Handbuch haben dann senden Sie diese bitte an Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences beh lt sich das Recht vor die Spezifikationen ohne vorherge hende Ank ndigung zu ndern Garantie et responsabilit Amersham Biosciences garantit l utilisa teur 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 livraison n est val able que si le produit a t install et utilis con form ment aux instructions fournies par Amersham Biosciences La soci t Amersham Biosciences ne sera en aucun cas responsable de tout dommage caus directement ou indirectement par toute utilisa tion incorrecte ou non approuv e du produit ou d coulant de cette utilisation y compris toute perte de b n fice ou de recettes toute perte de perspectives 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 Amersham Biosciences 1998 Tous droits r serv s La reproduction le stockage dans un syst me de r cup 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 stricte
6. RNA is usually well below 196 of that produced by the same concentration of DNA Solutions Important Refer to the material safety data sheet MSDS accompanying each chemical for detailed handling and safety information Hoechst 33258 stock dye solution 1 mg ml Hazard H 33258 is a possible mutagen Wear gloves and a mask and work under a fume hood 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 4 2 10X TNE buffer stock solution 100 mM Tris 10 mM EDTA 2 M NaCl 1000 ml Tris base Tris hydroxymethyl aminomethane MW 121 14 12 11g EDTA disodium salt dihydrate MW 372 20 3 729 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 pg 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 fil ter once dye has been ad
7. a second sam ple for each value sample 2 and average the two readings Note The 200 nM standard should display about 2000 fluorescence units Worksheet B 4MU standard measurements Carbonate luM 4MU Sample conc Reading Reading Avg reading buffer stock B x sample 1 sample2 y vol ml vol ul nM samples 142 2 2 0 1 9 1 8 1 7 5 3 Plot the 4MU concentration x vs the averaged readings y The result ing graph should be linear as shown in Figure 3 See page 4 8 for meth ods to analyze the data Figure 3 4 MU standard curve Fluorescence increases linearly with concentration 2000 1500 7 1000 Fluorescence intensity 500 0 0 50 100 150 200 Methylumbelliferone nM 5 4 Care and Maintenance To clean the exterior wipe the unit with a damp cloth Never use abra sive cleansers or 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 Important Turn the mains power off and unplug the power cord The optical surfaces are easily scratched Handle with extreme care and polish gently Wear gloves when servicing the optical block This pro tects both the technician from hazardous materials that may have been spilled and pr
8. 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 5 Analyze the results If following the protocols as described the actual concentration is dis played automatically If deviating from the protocols by using different concentrations or a calibration value that is a factor of the standard con centration use mathematical tools such as graphing and linear regres sion analysis to determine the concentration Plot standard curve measurements to confirm linearity in the range of interest 2 2 Important DyNA Quant 200 measurement notes 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
9. calculations for the low range assay amount added 2 ul x 100 ng ul 200 ng final concentration 200 ng 2 ml 100 ng ml 4 7 Analyze the results 1 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 2 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 2 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 0 Statistical analysis of the error in the fit gives a correlation coeffi cient r2 a measure of confidence in the data If the measured values near one end of the range deviate consistently from the best fit straight line the assay is being extended into a non lin ear region Samples should be diluted or assay conditions adjusted to retum 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 indi cation that the assay m
10. emission filter should slide out easily If required press the fil ter from behind with a cotton swab Cleaning Clean the cuvette well with cotton swabs Dampen a soft cloth with alcohol and wipe each optical surface If required gently polish with a dry soft cloth Remove all particles Allow to air dry All surfaces must be completely clean for accurate measurements Figure 4 Reference mirror Step 4 Optical block assembly N excitation aperture glass cover Step 3 y y d X Optical block bottom view Sample mirror Step 5 N LM __ Optical block Cuvette well top view Seal ring Reference mirror Emission ter Arrow points away from optical block Seal ring Emission filter d Thumb screw Ground plate 6 3 Optical block assembly 1 If the reference mirror was removed chose the best surface to face the reference beam Slide the mirror into the slot until it stops The mirror self aligns 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 mirror must fit flush with the top of the block 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
11. sea la causa que se deban a la uti lizaci n defectuosa e incorrecta del producto Amersham Biosciences 1998 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 Francais Deutsch Renseignements importants d utilization Pour une bonne compr hension et une utilisa tion en s curit maximale il convient de lire enti rement ce manuel Dans la documentation qui accompagne l instru ment un point d exclamation dans un triangle quilat ral a pour but d attirer l attention de l utilisateur sur des instructions importantes de fonction nement ou de maintenance Tous vos commentaires sur ce manuel seront les bienvenus et veuillez les adresser Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences se r serve le droit d effectuer des modifications de ces sp cifications sans aucun pr avis Wichtige Benutzerinformationen F r ein vollstandiges Verstandnis und eine sichere Handhabung dieses Produktes ist es notwendig da der Benutzer dieses Handbuch vollstandig durchliest Ein Ausrufezeichen in einem gleich seitigen Dreieck soll den Benutzer auf die Anwesenheit wichtiger Betriebs und Wartungsanweisungen in der dem Ger t beiliegenden Dokumentation hinweisen Wenn Sie
12. 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 measure ment Rinse Drain the cuvette completely by blotting while inverted on a paper towel 2 3 Operating Instructions This section describes instrument operation For DNA quantitation pro tocols 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 Numeric keys 0 9 SE Use the numeric keypad to enter a calibration standard RAC value or to choose menu FLUOROMETER options amp The lt ESC gt key displays the Q e Main Menu S The lt ENTER gt key registers numeric values advances to the next screen or initiates a fluorescence measurement o The lt SEND gt key sends The lt CALIB gt key calibrates The lt ZERO gt key sample ID number and the instrument based on the subtracts the assay displayed reading to the standard solution provided background serial communication port 3 1 Power up and program flow Turn the mains power switch beside the power cord receptacle to on to activate a self diagnostic 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
13. the stan dard 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 B 2 Figure 6 Effect of AT on relative fluorescence 2000 1750 1500 L 1250 1000 Relative Fluorescence 750 500 250 0 te T l 1000 ng ml 100 ng ml ege 40 60 80 DNA composition AT96 100 DNA samples of Microccocus lysodeikticus AT 23 E coli strand B AT96 50 calf thymus AT 58 Clostridium perfrin gens AT 69 and Poly dA Poly dT AT 100 all Sigma were dissolved in TNE buffer Initial concentrations were determined 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 E DNA 1000 ng ml H 33258 1 0 ug ml DNA 100 ng ml H 33258 0 1 ug ml An example using calf thymus DNA as a standard for fluorescent F 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 6sq AT samp 100 for 100 ng ml standard Adjusted standard setting 2 5 58 50
14. times place cuvette in well close the lid and press lt CALIB gt Enter the actual concentration of the standard Low range assay calibration value 100 ng ml High range assay calibration value 1000 ng ml Or enter a convenient value that will display a multiple of the actual DNA concentration Press ENTER 4 5 4 6 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 corresponds 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 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 fluores cence Dotsflash in the lower left corner until the measurement stabilizes Record the displayed value Drain the cuvette If desired repeat the measurement wit
15. to a fondo per soddisfare i requisiti specificati La garanzia inclusa nelle condizioni di consegna risulta valida solamente se il prodotto stato installato ed utilizzato nel rispetto delle istruzioni fornite da Amersham Biosciences Amersham Biosciences non potr essere ritenuta responsabile di incidenti o danni conse quenziali inclusi ma non limitati a perdite di profitti mancato guadagno perdite di affari difet ti di funzionamento e relative esposizioni dovuti ad un utilizzo non corretto del prodotto Amersham Biosciences 1998 Tutti i diritti riservati Nessuna parte della pre sente pubblicazione pu essere riprodotta conser vata in sistemi di gestione dati o trasmessa in alcun forma n per nessuno scopo senza autoriz zazione scritta del produttore Garant a y responsabilidad Amersham Biosciences garantiza que el producto entregado ha sido probado a fondo para comprobar el cumplimiento de las especifica ciones publicadas La garant a incluida en las condiciones de entrega s lo es v lida si el produc to se ha instalado y utilizado de acuerdo con las instrucciones entregadas por Amersham Biosciences Amersham Biosciences no ser respons able bajo ning n concepto de da os directos o indirectos incluyendo sin limitaci n la p rdida de beneficios la p rdida de ingresos la p rdida de oportunidades de negocio la p rdida de uti lizaci n y otras consecuencias relacionadas cualquiera que
16. 100 2 5 8 100 220 100 2120 Set the DyNA Quant Fluorometer to display 120 when measuring your 100 ng ml calf thymus DNA standard to read E coli DNA concentrations in ng ml B 3 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 microfluormometric DNA determina tion 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 conformation Anal Biochem 179 401 403 1989 Gallagher S In Current Protocols in Molecular Biology F A 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 Anal 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
17. 8 mg Distilled water to 100 ml Store at 4 C away from light 4MU stock solution B 1 uM 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 MW 105 99 21 29 Double distilled water to 1000 ml 5 1 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 deter mined The following steps assume the prompt is Off If unfamiliar with the instrument select Prompt on by choosing 2 Setup from the Main Menu and then pressing 1 Since the concentration is not measured in ng ml select no units under 2 Setup from the Main Menu 2 Units 2 None 1 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 ori entation close the lid and press ZERO 2 Calibrate the unit Add 100 ul of stock solution B 1 uM 4MU mix by pipetting into a dis posable transfer pipette several times close the lid and press lt CALIB gt Enter 500 within 10 seconds because this solution photodegrades quickly Press ENTER The fluorometer will now display 500
18. Capillary tubes 100 ul 100 80 6228 01 D 1 Capillary Cuvette Capillary Cuvette Adaptor Kit 1 Includes one capillary cuvette capillary tubes 9 ul pkg of 250 and 5 64 Allen key Capillary tubes 9 ul glass 250 Dye and standards Hoechst 33258 dye 100 mg 1 Calf thymus DNA standard 250 ug 4 methylumbelliferone standard 100 mg 1 Performance Validation Kit 1 Accessories Repipet Jr dispenser adjustable from 0 2 0 ml in 0 1 ml divisions 1 mounted on an 8 oz amber glass bottle DyNA Quant 200 Application Notes Protease Assay B Galactosidase Assay B Glucuronidase Assay D B Hydroxybutyrate BHB NADH Coupled Assay Fluorescent Probe Studies of Proteins Fluorescence Assay for DNA Quantitation Fluorescence Quantitation of PCR Products Before and After EasyPrep Purification using the DyNA Quant 200 Fluorescence Quantitation of Commonly Used Plasmid DNAs Using Calf Thymus DNA as a Calibration Standard 9 Fluorescence Quantitation of Double Stranded DNA after cDNA Synthesis 10 Fluorescence Quantitation of PCR Products Using the DyNA Quant 200 Prior to Re Amplification and Direct Sequencing 11 Fluorescence Quantitation of Single Stranded M13 DNA S BEEREBSE Printed in the USA D 2 80 6228 39 80 6228 58 80 6226 87 80 6227 06 80 6227 25 80 6252 52 80 6228 77 80 6236 37 80 6236 56 80 6236 75 80 6236 94 80 6237 13 80 6240 74 80 6329 09 80 6323 58 80 6333 46 80 6338 59 80 6370 89
19. Fluorometer Function and Description 11 Unpacking Se us CERES VERGI LP ee AE E Ee gd 1 1 Specifications cesses m Rr AR AD pec tU EYE 1 3 Important Information 1 4 Instrument set up n 1 5 Fluorometry Principles and Method Overview Fluorescence Measurement 2 1 Method overview 2 2 Important measurement notes 2 3 Operating Instructions User interface maras 3 1 Power up and program Tow 0 00002 ee eee 3 2 Main Men io ae e epe pp Lica aig da eoe et peer 3 3 Error and other messages 3 8 DyNA Quant communications with other devices 3 10 DNA Quantitation Guidelines for H33258 DNA ae 4 1 SOIULIONS sek A RSs Send AOL MPREt fap 4 2 Protocol sn id pe Reb EP 4 4 Generate a standard concentration CUVE o a aoaaa auauai 4 7 Analyze the results liiis 4 8 Enzyme Activity Quantitation SOIULIONS e rrea etr ep Ro Seen e a ease sets 5 1 Protocol f nos asa oium on e et Se ae Sn AOA Zu END Ness 5 2 Generate a standard concentration CUVE o on aaua aoaaa 5 3 Care and Maintenance Cleaning eet ettet ep pet e ee HN ted 6 1 Optical block disassembly iiiiisiisiss isis esses 6 2 Optical block assembly 6 2 7 Troubleshooting sss 7 1 Appendices A Enzyme assay protocols B Glucuronidase iioii eee A 1 B Galactosidase o poaren a eee A 7 B Effect of A T content on fluorescent DNA quantitation B 1 Bibliography ooooooocococcc eee C 1 Customer S
20. Fluorometer Function and Description The Hoefer DyNA Quant 200 Fluorometer is a filter fluorescence pho tometer with a fixed excitation bandpass source 365 nm and an emis sion bandpass filter 460 nm It is designed specifically for the accurate quantitation of low DNA concentrations using Hoechst 33258 dye The instrument can also measure enzyme activity based on the cleavage of coumarin methylumbelliferyl linked substrates as well as other fluores cent assays for which these excitation and emission wavelengths are appropriate Please note Fluorescent emission output is not strictly linear and it is affected by numerous variables If the procedures in this manual are fol lowed closely accurate concentration measurements can be made with a high degree of reliability A calf thymus DNA standard and the Hoechst 33258 dye are included to provide a reference point to calibrate the instrument The sample mea surement 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 adaptor and capillary tubes for 10 to 100 ul 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 li
21. For 100 ml extraction buffer mix 1M NaHPO pH 7 0 5 00 ml 2 mercaptoethanol 0 07 ml 0 5 M Na EDTA pH 8 0 2 00 ml 30 Sarcosyl 0 33 ml 1096 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 B 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 Generate a concentration curve Follow the instructions in section 5 to calibrate the instrument and gen erate a concentration curve using the 4 MU standard This procedure demonstrates 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 sus pension 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 expression 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 lin
22. When this message displays 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 instrument place blank assay solution into the cuvette and press lt ZERO gt Blank value is higher than the sam ple value Zero using blank capil lary assay solution and recalibrate The entered calibration value is too high Recalibrate with standard solu tion Use a lower factor if not using the actual standard concentration Diagnostic test failed Call the Amersham Biosciences Technical Service Department 3 9 DyNA Quant 200 communication with other devices Communication between the DyNA Quant 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 1 5 into the DyNA Quant 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 SEND to transfer each reading to the device Software options If the data flow is channeled to a computer data can be captured by Terminal software included with Mi
23. aCI 125 mM 0 5 ml 0 1 M MgCl 2mM 0 4 ml 2 mercaptoethanol 12 mM 17 0 ul Distilled water 18 5 ml 4 MUG FW 338 3 0 3 mM 100 ul methylumbelliferyl B D galactoside Dissolve 2 mg into 100 ul abs ROH 0 596 of the total volume of cocktail and then rapidly vortex into the aqueous cocktail solution until dissolved TCA solution 2596 w v trichloroacetic acid Generate a concentration curve Follow the instructions in section 5 to calibrate the instrument and gen erate a concentration curve using the 4 MU standard This procedure demonstrates the linearity of readings in the expected range of the assay Assay 1 2 3 4 5 ON Add 40 pl of sample to a microcentrifuge tube Use H50 for a blank Add 160 ul of the reaction cocktail Incubate at 37 C for 30 minutes Stop the reaction by adding 50 ul of 25 TCA Cool on ice Clarify the solution by centrifugation in a microcentrifuge for 1 to 2 min utes Add 0 1 ml of supematant to 1 9 ml of glycine carbonate reagent Read the fluorescence Determine the concentration from the standard concentration curve of 0 to 200 nM 4MU Notes on the standard procedure 1 A 40 ul sample should contain 10 6 to 105 units nM 4MU min 1 of B galactosidase activity If other dilutions of the sample into the reaction cocktail are used adjust samples accordingly The glycine carbonate reagent is sufficient to titrate 1 3 its own volume in 5 TCA to proper pH for reading 200 ul
24. an genes can be analyzed by using fusion genes containing the promoter of interest attached to the bacteri al B galactosidase gene The level of B galactosidase expression indicates the level of transcription under different regulatory conditions Sensitive and quantitative assays of p galactosidase activity are often needed The following assay measures the hydrolysis of the fluorogenic B galactosidase substrate Cleavage of 4 methylumbelliferyl D D galacto side by p galactosidase yields the fluorescent molecule 4 methylumbellif erone 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 Materials DyNA Quant 200 Fluorometer glass cuvette Microcentrifuge A 7 Solutions Glycine carbonate stop buffer 1 liter Glycine 133 mM 75 19 NaCO 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 5 1 Prepare 50 nM 4MU standard solution just before use 1 uM 4MU solution 100 0 ul Glycine carbonate stop buffer 1 9 ml Reaction cocktail 20 ml Prepare the reaction cocktail minus substrate at room temperature The substrate 4 M UG is most easily dissolved in cocktail by first dispersing in absolute ethanol 1M Tris HCl pH 7 5 25 mM 0 5 ml 5M N
25. clean Slide the assembled optical block into the instrument and secure with the thumb screw 6 4 Troubleshooting Always be sure to V NNN 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 Fluorescence values drift V NNN S S 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 measure ment is taken to avoid heating and photobleaching destruc tion of the fluorescent compound by light Protect fluorescent reagents and samples from light to pre vent 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 present 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 asa particulate drifts in the light path and then drop as it moves out of the beam range
26. crosoft Windows To use the Windows Terminal program 1 Under a standard Windows setup the Terminal program resides in the Program Manager under Accessories Double click on the Terminal icon to open the program If no Terminal icon is available select Run under the File menu and type Terminal 2 Setthe connection settings from the M enu bar by choosing Settings and then Communications 3 Select the COM port connected to the DyNA Quant 200 cable Then set the Baud rate at 1200 Data bits at 8 Stop bits at 1 Parity at None and Flow control at None Click OK The program is ready to receive data 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 a cor rection factor that can be applied if the composition of the DNA standard used is dissimilar to the DNA sample Guidelines for the H 33258 DNA assay Factors that affect the assay 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 reference for most ani mal and plant DNA because it is double stranded highly polymerized and is approximately 4296 GC 58 AT A different standard may be required for specific types o
27. d adjusts the pH for quantitating 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 Materials Enzyme control Substrate Calibration standard Reagents Equipment A 2 B glucuronidase GUS liquid suspension from E coli Boehringer Mannheim 127 680 4 methylumbelliferyl B D glucuronide MW 2352 3 Clontech 8082 Molecular Probes M 1490 Boehringer Mannheim 270 954 Sigma Chemical Co M 9130 4 methylumbelliferone sodium salt MW 198 2 AMU 7 hydroxy 4 methylcoumarin B methylumbellifer one Na HPO4 MW 141 96 NaH PO4 MW 119 98 Na2CO3 MW 105 99 Na EDTA MW 37224 Sarcosyl 2 mercaptoethanol Triton X 100 DyNA Quant 200 Fluorometer glass cuvette If using disposable plastic cuvettes order fluorescence grade The 0 2 ml Hoefer RePipet Jr is useful for dispensing stop buffer Solutions 4 Methylumbelliferone standard 50 nM 4MU in carbonate stop buffer Prepare standard stock solutions and carbonate stop buffer as described on page 5 1 Prepare 50 nM 4MU standard solution just before use 1 uM 4MU solution 100 ul Carbonate stop buffer 1 9 ml GUS extraction buffer 50 mM NaHPO pH 7 0 10 mM 2 mercaptoethanol 10 mM NasEDTA 0 1 sodium lauryl sarcosine 0 196 Triton X 100
28. ded Assay solution B for the high range DNA assay 100 5000 ng ml final DNA conc 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 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 4 3 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 sam ples can be determined Chose the standard concentration range low or high according to the expected DNA concentration and calibrate with this standard dilution One reference 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 standard a relia bility 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 s
29. e diagnostic test failed Call the Amersham Biosciences Technical Service Department 7 4 p 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 Jefferson and co workers Jefferson 1989 and is now commercially avail able from Clontech Laboratories in a variety of configurations The recombinant GUS gene mRNA and enzyme can be routinely manipulat ed and assayed to study promoter function tissue specific expression developmental regulation mRNA stability excision events of transpos able elements and signal sequences that target proteins for various organelles The advantages of using GUS to report the activity 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 variety of companies including Molecular Probes Research Organics and Clontech Typically GUS activity in solution is determined with the flu orogenic substrate 4 methylumbelliferyl B D glucuronide MUG MUG non fluorescent GUS glucuronic acid 4MU fluorescent ep The reaction product 4 methylumbelliferone 4MU is maximally fluo rescent at high pH where the hydroxyl group is ionized Addition of a basic solution of sodium carbonate simultaneously stops the assay an
30. ear 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 1 Dilute 10 ul of the commercial enzyme stock into 1 ml extraction buffer for a 1 100 dilution Further dilute this by adding 10 pl of the 1 100 dilu tion 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 2 Totwo 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 3 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 5 Foreach time point transfer 50 ul aliquots from each test tube staggered by 30 seconds into 2 ml stop buffer to quench the reaction 6 Read sample fluorescence A typical time course assay result is shown in Figure 5 Figure 5 Typical time course assay results 600 500 400 300 200 Relative Fluorescence units Time min
31. ed in Section 4 and the GUS assay is described in Section 5 For a current list of DyNA Quant 200 Application Notes see Ordering Information in the Customer Service Information section of this manual 2 1 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 abbre viated guide to measure the concentration of an unknown sample 1 Prepare the appropriate standard assay and sample solutions See Section 4 for DNA quantitation and Section 5 for GUS assays 2 To change settings to different operator preferences Choose2 Setup to select options such as prompt mode concentra tion units and auto send 3 To calibrate the instrument Pres 1 Read Set the zero by inserting the blank cuvette contain ing the assay solution without standard or sample Close the lid Press lt ZERO gt After 0 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 4 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 solution and mix well Insert the cuvette into the well close the lid
32. er J The Operon Cold Spring Harbor Laboratory Cold Spring Harbor New York 1981 Ullman A Jacob F and Monod J Characterization by in vitro complementation of a pep tide corresponding to an operator proximal segment of the B galactosidase structural gene of Escherichia coli J Mol Biol 24 339 343 1967 A 10 Effect of A T content on fluores cent 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 concen tration 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 AT96 of the standard and the sample are similar no correction for the base composition is required So when measuring eukaryotic DNA which has an AT ranging from 56 to 60 the standard DNA is typi cally calf thymus with an AT96 of 5896 DNA standards with different AT96 are available and can be selected to match the characteristics of your sample A range of standards available from Sigma Chemical com pany are listed below Double stranded DNA source GC 96 Sigma Chemical Co Ultra pure catalog number Calf thymus 42 D 4764 Clostridium perfringens 26 5 D 5139 E coli 50 D 4889 Huma
33. ervice Information Technical Service and Renate D 1 Ordering Information D 2 Italiano Espafiol Informazioni importanti per l operatore Per un utilizzo sicuro del prodotto leggere attentamente l intero contenuto del presente manuale Il punto esclamativo all interno di un triangolo equilatero indica all operatore la pre senza di importanti istruzioni di fun zionamento e manutenzione nella documentazione allegata al prodotto Si prega di inviare eventuali commenti al presente manuale a Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences si riserva il diritto di apportare modifiche ai dati tecnici senza preavviso 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 ngu lo equil tero en el manual advierte al usuario sobre la presencia de instruc ciones importantes de operaci n y mantenimiento del aparato Si desearan hacer alg n comentario sobre este manual tengan la amabilidad de remitirlo a Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences se reserva el dere cho a modificar las especificaciones sin previo aviso Garanzia e responsabilit Amersham Biosciences garantisce che prima della consegna il prodotto stato collauda
34. f 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 range 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 compared to 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 Refer to DNA Quant 200 Application Notes 8 and 11 listed in the order ing information section for more detailed information 4 1 Factors with little or no effect Buffers commonly used to extract DNA from whole cells Low levels of detergent 0 0196 SDS Generally the final detergent con centration should be well below the detergent critical micelle concen tration 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 concentrations 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 fluores cence due to
35. fluores cence 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 pretreat ing 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 report ed to lower the background Moe et al 1994 7 3 Error and other messages One dot remains in the left corner 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 Turning lamp on y The lamp was inadvertently turned off or the auto shut func tion switched the lamp off after one hour of no keypad activi ty This message reports that the lamp is being turned on Wait 15 minutes before taking measurements to allow the lamp to stabilize Zero first using a blank sample y Assay solution was not blanked Zero the instrument place blank assay solution into cuvette and press ZERO Blank gt sample Zero and re calib Blank value is higher than the sample value Zero using blank capillary assay solution and recalibrate Re calib using lower value Y The entered calibration value is too high Recalibrate with standard solution Use a lower factor if not using the actual standard concentration Failed Th
36. for a 50 nM solution of 4MU 2 0 ml of a 1 nM solution equals 2 pmol of AMU 3 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 reproducible Empty the cuvette between each measurement and rinse Drain the cuvette com pletely by blotting it while inverted on a paper towel Note If the reading exceeds the display range 25000 dilute the sample Once the initial reference value can be reliably reproduced proceed to determine concentrations of unknown samples or determine assay linear ity 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 5 2 Generate a standard dilution curve Example Determine measurements for the series of concentrations in Worksheet B below Add the appropriate amount of carbonate stop buffer and place the cuvette in the cuvette well Close the lid and zero the instrument Remove the cuvette and add the corresponding volume of standard Mix thoroughly and place the cuvette into the well in its original orienta tion close the lid and record the display value Measure
37. h a second sample and average the readings If the unknown sample readings exceed the display range 25000 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 concentration 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 concentrations 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 stan dard 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 two readings Worksheet A Low range standard measurements DNA standard Reading Reading Avg reading y conc x vol ul sample 1 sample 2 samples 142 2 ng ml 0 100 200 300 400 500 10 100 ng ul DNA to be added to 2 ml assay solution A Standard curve volume
38. ight be out of the linear range Figure 2 Concentration curve using calf thymus DNA for low range assay 600 Data analysis example e Duplicate data points were plotted and analyzed by a lin 400 ear least squares regression Best fit parameters 300 r 0 9998 b 0 95 m 1 00 y 1 00x 0 95 200 4 Fluorescence units 100 4 To find an unknown DNA concentration assign the dis 0 e play value to the y variable 0 400 200 300 400 500 600 and solve for the DNA con centration x DNA concentration in the cuvette ng ml 4 8 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 Specific protocols for assaying B galactosidase and B glucuronidase activ ities are in Appendix A The following additional protocols are available upon request Application Note 1 Protease Assay Application Note 4 D p Hydroxybutyrate BHB NADH Coupled Assay Application Note 5 Fluorescent Probe Studies of Proteins Solutions 4MU stock solution A 1 mM 100 ml 4 methylumbelliferone sodium salt MW 198 20 19
39. its 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 orientation 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 3 5 Main menu option 2 gt Setup Select 2 from the Main Menu to access the Setup menu which accepts operator preferences 1 gt Prompt 3 gt Send 2 gt Units 4 gt More 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 Prompt 1 gt 0ff 2 gt 0n Units 1 gt ng ml 2 gt None 1 gt Manual send 2 gt Auto send 3 6 1 gt Prompt off displays only mea surements and minimal instruc
40. n placenta 42 D 4642 Micrococcus luteus 72 D 5014 B 1 If measuring samples which differ significantly in AT96 from the selected DNA standard a modification to the calibration protocol is needed As shown in Figure 6 fluorescence is a linear function of AT96 when the DNA concentration based on A eo is held constant This relationship was based on several DNA samples with AT ranging from 23 to 100 measured with the DyNA Quant 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 sug gesting 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 AT96 effect and DNA concentration effect are both linear it is straightforward to calibrate the assay when the AT of the stan dard 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 a different AT Adjusted Standard Setting Card 0 025 AT AT 6samp 1 The adjusted setting is the calibration to use for the standard at the C 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 AT96 of 40 and 50 respectively and
41. nd drain Add only assay solution no sample to cuvette Place cuvette in well always in the same orientation and zero the instrument as before Remove cuvette add sample and mix Place cuvette in well close lid and record the mea surement Displays fluorescence in chosen units Press lt ENTER gt to re read the sample concentration N V o o e o o a Measurement eeeeeececce e zs Instrument calibration Prompt on If prompt on is selected each step displays as follows Press the indicated key or press ENTER to continue LCD message 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 Calib ESC gt Main Menu Action required 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 un
42. olution mixed with 2 ml assay solu tion is a 1000 ng ml standard solution The calf thymus standard is supplied in dry form Follow the instructions accompanying the standard precisely to achieve the proper dilution 4 4 Important notes Accurate pipetting and thorough mixing are critical for reproducible results use a micropipetter accurate to 0 02 ul Orient the cuvette the same way each time you place it in the sample cham ber 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 instrument is stabilizing a measurement If one dot remains in the left corner check the Troubleshooting section 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 On 1 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 displays 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
43. otects the optical surfaces from fingerprints Use only isopropanol on a clean soft cloth to clean optical surfaces 6 1 Optical block disassembly 1 Turn the mains power off and unplug the power cord Spread a soft cloth over the work area and tum the unit upside down onto the padded sur face Wear gloves both to protect yourself and the optical surfaces Locate the thumb screw near the front of the unit and unscrew The cap tive thumbscrew stays attached to the block Lift the optical block assem bly straight up Hold the optical block assembly so that the ground plate with the thumb screw faces up and the optical block is cradled in your palm In this posi tion 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 aper ture Keep the optical block in this position for steps 4 and 5 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 nudged 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
44. prepared with acid guanidinium thiocyanate phenol solution V Fluorescence of lysates prepared without an alkaline EDTA pretreatment is reduced by 7096 compared to lysates with such pretreatment Alkaline conditions allow formation of complexes between DNA and the dye For a detailed protocol see Rymaszewski et al 1990 Estimation of cellular DNA content in cell lysates suitable for RNA isolation Anal Biochem 188 91 96 Use the appropriate reference standard V V Y 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 lin ear may have slightly different dye binding characteristics Readings higher than expected V Fluorescence enhancement may result from high levels of detergents Final SDS concentration should be below 0 0196 and other detergents below 10 ug ml the final concentration of any detergent should be well below its critical micelle con centration Final Triton X 100 conc must be below 0 00196 Use a standard with a G C content very similar to that of your sample Single stranded genomic DNA yields about half the
45. r the instrument when it is p used in laboratory locations and p used as delivered from Amersham Biosciences except for alterations described in the User M anual 1 3 j Important information 1 4 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 so 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 acces
46. reaction stopped with 50 ul of 25 TCA The protocol is designed for assay of F coli B galactosidase activity which is active at neutral pH The vertebrate form of p galactosidase is a lysoso mal 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 Depending on the calibration setting used maximum readings are obtained at 40 to 200 nM 4MU final concentration in the glycine car bonate buffer Limitations on the sensitivity of the assay are determined by the back ground fluorescence of the substrate It is therefore important to use freshly prepared substrate solutions in assays where high sensitivity is desired Frozen reaction cocktails may be used but backgrounds gradually increase with repeated freeze thaw cycles Substrate sometimes precipi tates 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 Expression of Bacterial B 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 Mill
47. sories and parts approved or supplied by Biosciences may be used for operating maintaining and servicing this product Instrument set up Mains power 1 2 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 Tum the mains power switch beside the power cord receptacle to on 1 3 See section 3 for complete operating instructions Serial port connector The RS232C serial port is a DB9 9 pin male connector The type of serial cable required depends on the type of device DTE or DCE that it is connected to The DyNA Quant is configured as a DTE device so a con nection to another DTE devices 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 DyNA Quant RS232C signal and pin number assignments Pin 2 Transmit Pin 3 Receive Pin 5 Ground Other pins Not connected The DyNA Quant requires these settings in the device receiving data Baud rate 1200 Data bits 8 Stop bit 1 Start bit 1 Parity None 1 5 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 exci tation spectr
48. st 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 con tact the carrier immediately Be sure to keep all packing material for dam age claims or for repacking should it become necessary to return the unit 1 1 Figure 1 Main components The power switch power cord recep tacle and communications port are on the rear panel access the cuvette well by pressing the lid release 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 I Included but not shown Q J Calf thymus DNA standard Hoechst 33258 dye 1 2 Specifications Power input rating Fuse value Light source Lamp output Emission filter Environmental operating conditions Installation category Pollution degree Dimensions Product certifications 115 V or 230 V 47 63 Hz T 3 15A 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 II 2 13 x 16 5 x 35 cm hxw xd UL3101 1 CSA C22 2 1010 1 CE This declaration of conformity is only valid fo
49. the instrument is ready Amersham Biosciences DyNA Quant 200 System Diagnostic A series of screens report which components are being tested as the pro gram runs through the system diagnostics Finally a 20 second count down appears as the system warms up The Main Menu appears when the instrument is ready to receive input 1 gt Read 2 gt Setup 3 gt Test Important Allow 15 minutes for the lamp to stabilize before taking any measurements 3 2 Main Menu The Main Menu accesses the three different functions 1 gt Read mea sures fluorescence 2 gt Setup sets operator preferences and 3 gt Test runs a comprehensive diagnostic routine 1 gt Read 2 gt Setup 3 gt Test Press lt ESC gt anytime to return to this screen The following three sec tions 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 The read option prepares the instrument to measure fluorescence This function can be accomplished with either Prompt off which is the default setting and does not guide the operator at each step or the oper ator can choose Prompt on which describes each step of the DNA assay Select from the 2 gt Setup menu The display for each option is full
50. 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 3 7 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 Data 3 gt Info 2 gt Lamp 4 gt Diagnos 3 8 1 The Data option displays volt age mV signals from the sample Sig and the lamp reference Ref 2 The Lamp option switches the lamp off if it is on or on if it is off 3 TheInfo option identifies the initial UV lamp reference signal mV the Firmware version the PC board version the date of manufac ture and the serial number 4 The Diagnos option initiates a comprehensive 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 Turning lamp on Zero first using a blank sample Blank gt sample Zero and re calib Re calib using lower value Failed This message reports that the lamp is being switched on Wait 15 min utes to allow the lamp to stabilize before taking measurements
51. tions 2 Prompt on guides the user through the assay step by step 1 Sets units to display ng ml 2 No units will be displayed After a brief pause the Setup menu displays 1 The Manual send option sets the software so that measurements and the corresponding ID numbers are transmitted to the serial port only when SEND is pressed 2 The Auto send option conveys this data automatically after each measurement See pages 1 5 and 3 10 for printer connection guide lines After a brief pause the Setup menu displays 5 gt Autoshut 6 gt ID 7 gt Language Lamp auto shut 1 gt 0f f 2 gt 0n Please enter ID number 0 1 gt Engl 2 gt Deutch 3 gt Franc 4 gt Espan Press 4 gt in the Setup menu for these additional options 1 gt Lamp auto shut of f causes the lamp to stay on until the instrument is switched off That is the automat ic shut off is disabled 2 Lamp auto shut on causes the lamp to automatically 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 displays 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
52. u have any comments on this manual we will be pleased to receive them at Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences reserves the right to make changes in the specifications without prior notice Warranty and Liability Amersham Biosciences 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 Amersham Biosciences Amersham Biosciences shall in no event be liable for incidental or consequential damages including without limitation lost profits loss of income loss of business opportunities loss of use and other related exposures however caused aris ing from the faulty and incorrect use of the prod uct Amersham Biosciences 1998 All rights reserved No part of this publication may be reproduced stored in a retrieval system of transmitted in any form by any means without permission in written form from the company DyNA Quant 200 Fluorometer User M anual Amersham 80 6231 24 DQ200 IM Rev C1 5 98 e e Biosciences
53. um 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 the the photodetector allows only fluorescence at 460 nm 15 nm to register Thus the measured fluorescence is a direct indicator of the DNA concentration zu 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 The fluorescent B glucuronidase GUS assay does not depend on the for mation of a complex Instead measured fluorescence indicates the amount of reaction product 4 methylumbelliferone 4MU that is released by the GUS hydrolysis of 4MU glucuronide The same excitation and emission wavelengths apply however the shorter 365 nm wave length light excites the fluorescent 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 discuss
54. y described in the following two pages Once you are familiar with the instrument you will probably choose prompt off for routine measurements 3 3 Prompt off Prompt off is the default setting In this mode the operator is not prompted to zero every assay solution 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 3 4 Action required Note Pressing the enter key at every step is not required as it isin Prompt mode on Displays fluorescence in chosen units To zero the instrument Add only assay solution no stan dard 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 standard Press lt ENTER gt Displays fluorescence in the chosen units To measure fluorescence Remove the cuvette rinse a

1 Fluorometer Function and Description Unpacking

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