Pharmacia U2000
Contents
1. rae in Background Y N background lt Enter gt Set Reference H set ref fun gt 28 00 C RNA 1 RNA concentration ug ml AllV rat 1 900 RNA 1 A260 A280 ratio ae frode gt OLIGO Factor 33 000 Factor Background YIN background lt gt enter gt Set Reference EE Set ref fun 15 00 COLI 1 Oligo concentration ug ml AIM rat 1 600 OLI 1 A260 A280ratio DNA USING A SINGLE CELL HOLDER ACCESSORY A similar format is used for RNA and Oligo E ow Insert Reference Background Y N set Reference H setref 31 00C DNA 1 DNA concentration ug ml 14 English rat 1 750 DNA 1 A260 A280 ratio Issue 05 01 2000 PROTEIN IMPURITY Protein Concentration in Nucleic Acids For estimation of protein concentration in Nucleic Acid solutions one can automatically calculate the protein based on an equation derived by Warburg and Christian for crystalline yeast enolase This uses absorbances at 280 and 260 nm and is valid provided tyrosine and phenylalanine are present Protein mg ml 1 55 Abs 0 76 Abs 2280 2260 It is possible to calculate the coefficients in order to customise the equation for a particular protein by measuring absorbances at known concentrations for both 260 and 280 nm this generates a series of simultaneous equations which need to be solved for x and y the required coefficients In cases where x is calculated to have a negative coef2. This is displayed and set ref is pressed to start making measurements sample number and cell position are automatically incremented after measurement to stop making measurements or return to Absorbance mode increments sample number and cell position if cell changer is fitted The above and following sequences of instructions apply to the 6 cell changer which is supplied with the instrument There are different self explanatory displays if a single cell holder or sipper is installed and can be put in sequentially if a 6 cell changer is fitted Issue 05 01 2000 English There are two concentration modes Factor and Standard Factor Concentration mode is used when a conversion factor is known this is required to convert the absorbance measurement for a sample at a specific wavelength to a concentration by a simple multiplication of absorbance x factor Standard Concentration mode is used when a sample of known concentration is available by measuring the absorbance of this at a specific wavelength the conversion factor is calculated see above and this can be applied to other samples of unknown concentration This is equivalent to a one point calibration and assumes that a sample of zero concentration has zero absorbance FACTOR CONCENTRATION Concentration of unknown Absorbance x factor mode FACTOR CONCENTRATION Set Wave 360 seta 10 00 Factor 0 01 lt 9
3. Accessory 8 cell changer Select automatic reference Select automatic reference Accommodates cells 10 50mm Requires a circulating bath pathlength Locate round extension of tube restrainer into top of cell changer thumb screw O rings are to keep tubing together Replace front blanking plug with that supplied after threading tubes through it 6 position Peltier heated cell changer Spare 8 position cell changer 80 2106 04 80 2108 01 Accessory 6 cell changer Accessory 8 cell changer Select automatic reference Select automatic reference Requires Temperature Control Unit 80 2105 49 Cell compartment socket 1 28 English Issue 05 01 2000 Single Cell HolderAccessones Install by removing existing cell holder replacing with the baseplate plug supplied with the accessory mounting so that the finger locks push backwards and the arrow is on the front face followed by sample sample Cell Holder 10mm pathlength Cell Holder 50mm pathlength 80 2106 05 80 2106 07 Accessory Single cell holder Accessory Single cell holder Ultramicrovolume Cell Holder Microvolume Cell Holder 80 2106 06 80 2106 09 Accessory Single cell holder Accessory Single cell holder Use with Sul cell 80 2103 68 and Use with 50ul cell 80 2076 38 70ul cell 80 2103 69 Use 2 axis adjust screws to obtain maximum light throughput Issue 05 01 2000 English
4. H Indicates total instrument usage in hours BASELINE 19960102 Indicates date last baseline was run saved or not DATE 19960102 Set current date in year month day TIME 11 16 32 Set current time in hours minutes seconds LAB NAME These are printed out if GLP PRINT OUT is OPERATOR set to Yes for you to complete manually Refer INSTRUMENT to Appendix if you wish to enter your details for printing out page 41 Set up refers to instrument operating parameters and include facilities for switching lamps off running a baseline setting the instrument to work in GLP mode having automatic print on pressing RUN etc These would normally be set up one at a time on different occasions and once changed the instrument reverts to its default state Absorbance measurement mode Set up has battery back up UV LAMP Switch deuterium lamp on or off NEW BASELINE Create a temporary baseline to correct for the wavelength energy profile of the spectrophotometers light source s and reference if required Ensure lamps are warmed up 30 minutes for deuterium A temporary baseline is lost at instrument power down unless saved SAVE BASELINE Save a temporary baseline to permanent memory This can be the temporary baseline created above VIS LAMP Switch tungsten lamp on or off Issue 05 01 2000 English 23 UV LAMP SAVE Cause instrument to power up and calibrate as usual but then switch off the deuterium lamp automatically infrequ
5. display works see OPERATION The instrument is delivered with a stored baseline This is required to correct for the wavelength energy profile of the light source A new baseline should be stored when a lamp is changed or if the instrument is not used for a long time several weeks refer to Maintenance for details To enter laboratory name operator name and or instrument asset number details at this stage refer to Appendix Entry of alphanumeric characters for print out If this equipment is used in a manner not specified or in environmental conditions not appropriate for safe operation the protection provided by the equipment may be impaired and instrument warranty withdrawn 2 English Issue 05 01 2000 Essential Safety Notes There are a number of warning labels and symbols on your instrument These are there to inform you where potential danger exists or particular caution is required Before commencing installation please take time to familiarise yourself with these symbols and their meaning Caution refer to accompanying documents Background colour yellow symbol and outline black Z WARNING WARNING U V RADIATION IS HARM FUL TO YOUR EYES U V RADIATION IF POWER IS RESTORED WITH HOT TOP COVER REMOVED EYE PROTECTION MUST BE WORN Accessories Care should be taken when handling all heated accessories Ensure that the cell compartment lid is closed when operating cell changers a
6. if you forget the number contact your local Technical Specialist RESETS LAMP LIFE Resets UV and or visible lamp life to 0 hours when a new lamp is fitted and creates new baseline It also causes a reset of the maximum lamp energy for GLP Service Available for service engineer access only Returns instrument to normal operating condition with new setup parameters in lace 24 English Issue 05 01 2000 PrintOut Your instrument will print out in 40 character format on any centronics compatible parallel printer connected via the appropriate cable to the printer port on the rear panel simply press to print current display a Print instrument display every time it is updated or a measurement is run gt Set up V RUN PRINT GM gt on of Example Absorbance Ratio DNA concentration 31 00 DNA 1 ratio rat 1 750 DNA 1 Le ig ae b Print a header at the start of an experiment for header print out on demand Example Absorbance Ratio DNA with GLP on Mode of use DNA concentration of DNA mode DNA concentration Abs 260nm 50 units are ug DNA ml DNA ratio Abs at 260nm Abs at 280nm GLP calibrated 19960917 at 15 12 GLP Calibration PASS Instrument status accepted Operator sonale ee ere eee Date 19960917 Time 12 03 50 260 0 620 280 0 354 31 000 C DNA 2 rat 1 750 DNA 2 cell changer reset to cell 3 Lae ae Issue
7. local service engineer for advice During calibration if GLP PRINT OUT is on the instrument will wait 10 minutes to allow working temperatures to be reached before completing calibration The display will show Waiting for GLP press stop to abort this if required 40 English Issue 05 01 2000 Entry of alphanumeric characters for print out function Parameters V7 LAB NAME OPERATOR INSTRUMENT i When using the above utilities the nature of the keyboard keys changes A title 40 characters maximum made from an alphanumeric character string of upper and lower case characters numbers and punctuation marks may be entered abcdefghijklmnopqrstuvwxyz 1234567890 ABCDEFGHIJKLMNOPQRSTUVWXYZ lt gt 1 N amp _ space An individual character is chosen using the A v keys and selected using the PRINT key The gt key moves the cursor along the display A complete name or title is entered using the ENTER key lt gt AJLY V V setref V sample v print enter 300000 Move cursor along the display Move within the character string Move to start of character string Move way down character string to Move way down character string to Move way down character string to Delete the character at the cursor Select the character at the cursor Complete the alpha
8. or as change of absorbance or concentration per unit time interval DELTA DATA When DELTA DATA results are constant the reaction is proceeding at a linear rate RAW DATA results can be output to a chart recorder for a visual interpretation of the change with time both RAW and DELTA DATA can be numerically output to a parallel printer Note that when in Rate Modes data is sent as an ASCII stream to the serial interface this can be picked up by a PC with Windows installed Use the terminal emulator to pick up the datastream settings are 19200 Baud 1 stop bit 8 data bits 0 parity After the reaction is finished paste the results into Excel and parse into separate column using Data gt Parse command Issue 05 01 2000 English 17 RATE RAW DATA display of actual absorbance with time KNR Mode gt RATE RAW DATA Set Wave 600 setX Time unit sec min Seconds or Minutes lt gt Delay 0 Delay Time 0 lt 999 Interval 1 Time Interval 1 lt 99 Chart recorder YIN Use chart recorder KI enter gt IfYEs Chart post delay YIN eae E KID Chart off at end YIN eni ii ane 5 IfNO Factor 1 000 Factor Set reference YIN Setreference If Yes H 600 0 375 A KNR 1 600 0 365 A 0 03 18 i Lt factor updated Output to the Amersham Pharmacia Biotech REC 101 chart recorder is sy
9. 05 01 2000 English 25 Messages The spectrophotometer goes through a multi step calibration sequence If for any reason the calibration is not completed satisfactorily the messages displayed if not self explanatory are either as follows or relate to a fault condition which requires a service engineer from your local supplier Other messages are displayed when an accessory is installed Error Messages Display GLP Calibration Fail lpt VIS calibrated lpt UV calibrated Abs Non Linear GLP on Too much light Beam Blocked Printer Error No Printer 26 Possible Causes Visible region only calibrated UV region only calibrated Instrument not at working temperature Misalignment of filter quadrant Dirty Filters Too much light in sample compartment Not enough light getting to detector RUN PRINT is ON GLP PRINT OUT is ON printer is off line or out of paper No active printer English Remedy Refer to Appendix GLP Diagnostic Tests Check and replace UV Lamp if necessary Check and replace visible lamp if necessary Let instrument reach working temperature Contact service engineer Close lid properly and ensure baseplate plug is in place if relevant Check light beam is not blocked and cell compartment empty If using a sipper remove flowcell Check printer state Connect or switc
10. 29 Cylindrical Cell Holder up to 100mm pathlength 80 2106 10 Accessory Single cell holder HPLC Cell Holder 80 2106 11 E Accessory Single cell holder Flowcell volume is 8ul Pathlength is 2 5mm Replace front blanking plug with that supplied after leading the two tubes through it Electrical Cell Holder 80 2106 12 Accessory Electric cell Select temperature of OFF 25 30 37 C Cell compartment socket 2 30 English Water Heated Cell Holder 10 40mm pathlength 80 2106 08 Accessory Single cell holder Requires circulation bath Replace front blanking plug with that supplied after threading tubes through it Peltier Cell Holder 80 2106 13 Accessory Thermostat holder Set required Temp in range 20 49 C using keypad Cell compartment socket 2 Programmable Heated Cell Holder E ae 80 2106 14 D Accessory Single cell holder Temp range is 20 100 C Used for Tm studies of DNA and RNA denaturing Requires PC and Temperature Control Unit 80 2105 49 Cell compartment socket 1 Issue 05 01 2000 OtherAccessones Sipper 80 2106 15 The Sipper introduces either a sequence of samples for a single reading or a single sample for a series of readings into the spectrophotometer It is used together with a single 10mm cell holder either heated or non heated A flowcell together with PTFE tu
11. 5 months old AND have had less that 750 hours use Fuse Replacement 1 Switch off the instrument and disconnect the power supply cord The fuse holder can only be opened if the power supply plug has been removed and is located between the power input socket and the on off switch on the back panel of the instrument 2 Slide open the fuse holder by pulling at the notch 3 Place fuses 2A 5mm x 20mm FST into the fuse holder and slide back into position 4 Reconnect the power supply cord and switch on the instrument Fuses are not normally consumed in an instrument s lifetime If they blow repeatedly contact your supplier Issue 05 01 2000 English 37 Cleaning and GeneralCare External cleaning Switch off the instrument and disconnect the power cord Use a soft damp cloth Clean all external surfaces A mild liquid detergent may be used to remove stubborn marks Sample compartment spillages Switch off the instrument and disconnect the power cord The cell holders baseplate and sample compartment are coated in a chemical resistant finish Strong concentration of sample however may affect the surface and spillages should be dealt with immediately Observe all necessary precautions if dealing with samples or solvents which are hazardous There is a small drain hole in the sample compartment to allow excess liquid to drain away Liquids w
12. 9999 Set Reference in gt 360 1200 C FC 1 op 360 1200 A 1 To show Abs reading Factor 10 English Issue 05 01 2000 STANDARD CONC Concentration of unknown Absorbance of unknown x Concentration of standard standard Absorbance of standard Set A Mode gt STANDARD CONC Set Wave 360 Set Reference Standard 1 000 360 1 000 C SC 1 360 0 800 C Sc 1 unknown Ge The calculated factor cannot be displayed Issue 05 01 2000 English Concentration of standard enter Concentration of unknown relative to standard Nucleic Acids There are 5 modes for Nucleic Acid Quantification studies using absorbance measurements at 260 and 280nm Mode Use DNA DNA quantification and purity checking RNA RNA quantification and purity checking Oligo Oligonucleotide quantification and purity checking Protein Impurity Estimation of protein concentration in nucleic acid solutions Abs Ratio For general use and for DNA RNA and oligonucleotides if a dilution factor is to be included DNA RNA Oligo Nucleic Acid Quantification Concentration can be measured using absorbance at 260nm because it is well established that a solution of DNA or RNA with an optical density of 1 0 1 0 Absorbance Unit has a concentration of 50 or 40ug ml respectively in a 10mm pathlen
13. CONTENTS Page UDSDWWN 28 34 39 43 Issue 05 01 2000 Section Unpacking Postioning and Installation Essential Safety Notes Symbolsused in Manual Keyboard Display Panel OPERATION Introduction Measurementmodes Absorbance Concentration Nucleic Acids Reaction Rate Scan Transmission Instrument Utilities Print Out Messages ACCESSORIES MAINTENANCE APPENDIX SPECIFICATION English Unpacking Postioning and Installa tion Inspect the instrument for any signs of damage caused in transit If any damage inform your supplier immediately Ensure your proposed installation site conforms to the environmental conditions for safe operation Indoor use only Temperature 10 C to 40 C Maximum relative humidity 80 up to 31 C decreasing linearly to 50 at 40 C The instrument must be placed on a hard flat surface for example a laboratory bench or table which can take the instrument weight 13 kg such that air is allowed to circulate freely around the instrument Ensure that the cooling fan inlets and outlets are not obstructed position at least 2 inches from the wall This equipment must be connected to the power supply with the power cord supplied and MUST BE EARTHED GROUNDED It can be used on 100 240V supplies The instrument rear panel is shown on the inner back cover of this manual Switch on the instrument and check that the
14. Ends current programme Access to Set up and Parameters Output information on the display panel to a parallel printer 4 English Issue 05 01 2000 Display Pa nel The display panel has 20 characters divided up as indicated Default start up values are shown and represent a wavelength of 360nm in absorbance mode with cell position number 1 in the light path 3610 olofo Il b cl al si f a Wavelength Display of current wavelength in nm b Numeric Result Display of absorbance T concentration absorbance ratio result Unit of Measurement Display of numeric result unit A is Absorbance Units T is Transmission C is concentration units d Mode of Operation Display of instrument enhancement mode FC and SC are factor and standard concentration modes DNA RNA OLI are DNA RNA oligonucleotide quantification modes PRO is protein concentration mode RAT is absorbance ratio mode KNR is display of absorbance with time KND is display of change of absorbance with time SCN is scan to chart recorder e Temperature Indicator Display of temperature status if a peltier heating accessory is in use C is at temperature HI is over temperature or cooling down LO is under temperature or heating up f Sample Number Display of sample number Issue 05 01 2000 English O PERATION Intoduction Your UV Visible spectrophotometer is a simple to use microproc
15. ances Transmission Wavelength 546 Setar setref 560 10 00 T BOC remove blank m 546 80 00 T 1 Issue 05 01 2000 English 21 Instrument Utilities Settings which can be changed are stored until overwritten Select from the table below with ZISIAIv Select On Off and Y N Options with nter lt gt Set a new DATE or TIME with lt gt keya dn eo CE e 4090 V1 9 19960102 UV LAMP This is reserved Retum to UV LAMP LIFE 27 H NEW BASELINE forService absorbance VIS LAMP LIFE 27 H SAVE BASELINE Engineers mode TOTAL USE 27 H VIS LAMP BASELINE 19960102 UV LAMP SAVE DATE 19960102 SOUND TIME 11 16 32 RUN PRINT LAB NAME GLP PRINT OUT OPERATOR RE CALIBRATE INSTRUM ENT LAMP CHANGEAT LOCK UNLOCK RESET LAMP LIFE Dates are shown in year month day format 22 English Issue 05 01 2000 Parameters Instrument parameters is a reflection of both the current instrument internal parameters EPROM version lamp lives date of baseline and the user definable documentation time date laboratory operator and instrument names this latter part should normally require one time user access only All parameters have battery back up 4090 V1 9 19960102 EPROM version and date of software UV LAMP LIFE 27H Indicates deuterium lamp usage in hours VIS LAMP LIFE 27 H Indicates tungsten lamp usage in hours TOTAL USE 27
16. bing and pump tubing is supplied with the Sipper Separate user instructions are supplied with the accessory Temperature Control Unit 80 2105 49 This is required to control the 6 position Peltier heated cell changer 80 2106 04 and the Programmable Heated Cell Holder for Tm studies 80 2106 14 Separate user instructions are supplied with the accessory Printer Stand 80 2106 60 Spare Baseplate blanking plug 80 2106 18 Spare dust cover 80 2106 19 Cell holder for 100mm pathlength 80 2107 14 Cell holder for use with magnetic stirrer 80 2108 10 magnetic stirrer required Issue 05 01 2000 English 31 Lamps Consumablesand otheritems Tungsten halogen lamp Deuterium lamp Pump head tubes 6 for Sipper PTFE flowcell tubing with connectors Replacement flowcell including tubing Autosampler interface kit Chart recorder cable Recorder REC 101 single channel Serial interface cable for connection to PC D9 male instrument to D9 PC Serial interface cable for connection to PC D9 male instrument to D25 PC Centronics parallel printer interface cable 80 2106 16 80 2106 17 80 2080 74 80 2055 13 80 2080 60 80 2104 96 80 2105 95 18 1001 42 80 2105 97 80 2106 51 80 2071 87 Separate information giving details on serial and parallel interface connections if required is available from a Service Engineer with your local supplier whom you should contact for further details 32 English Issu
17. dards Certificated engineers and calibrated test equipment Approved to ISO 9001 standard Choice of agreement apart from break down coverage can include Preventative maintenance Certification When using calibration standard filters insert such that the flat surface is facing away from the spring end of the cell holder Observe all necessary precautions if dealing with hazardous samples or solvents User maintenance is restricted to changing the instrument lamps and mains fuse For any other maintenance operation contact your local supplier 34 English Issue 05 01 2000 Lamp Replacement Replacement lamps are available from your supplier using the following part numbers Deuterium lamp 80 2106 17 includes tungsten lamp as well Tungsten lamp 80 2106 16 The deuterium lamp is supplied fitted onto a plate a new tungsten lamp is also included Although deuterium lamps are covered by their own warranty an engineer s call out fee is not and users are advised to change their own lamps Lamp replacement is very easy and the process has been designed so that the user can do it without the need to call out a local service engineer No lamp alignment is required as the lamp select mirror is automatically aligned for maximum lamp energy throughput during the instrument calibration procedure The lamps become very hot in use Ensure they are cool before changing Do not touch the opt
18. e 05 01 2000 SWIFT II Applications Software SWIFT II comprises application modules for wavelength scanning reaction kinetics quantification multi wavelength time drive and fraction analysis and can be used to enhance the software already included on the spectrophotometer Specific application packages consist of groups of modules for general analytical purposes biochemistry and molecular biology SWIFT II LAB and for method development and quality control SWIFT II METHOD 80 2108 31 SWIFT II LAB for general analytical purposes Wavelength scanning Reaction Kinetics Quantification Time Drive 80 2108 31 SWIFT II METHOD for method development Wavelength scanning Reaction Kinetics Quantification Time Drive Multi Wavelength Fraction Analysis Recommended PC for proper operation For optimum performance an IBM compatible 486 or greater personal computer running Microsoft Windows 95 is required The PC should have a minimum of 8MB RAM 200Mb hard disk a 1 44 MB 3 5 inch floppy disk drive a serial mouse installed and free COMMS serial port and VGA graphics Any printer supported by Microsoft Windows 95 can be used Contact your supplier for further information Issue 05 01 2000 English 33 MAINTENANCE After sales support We supply support agreements which help you to fulfil the demands of regulatory guidelines concerning GLP GMP Calibration certification using filters traceable to international stan
19. e Factor 100 If using a 10mm pathlength cell for DNA quantification and 10 x dilution use Factor 500 Mode gt ABS RATIO Wave 1 260 4 9 Wave 2 491 42 Background YNI fey gt If YES Wave B 320 AB Factor 50 00 Factor Set Reference Set ref qu rat 1 750 RAT 1 Absorbance Ratio NIV TEE a 16 English Issue 05 01 2000 Reaction Rate Rate studies can be used to measure the change in absorbance of a sample with time a typical example is an enzyme kinetics reaction rate study at 340 nm In this the change in concentration of either one of the substrates or one of the products of the reaction is monitored at a particular wavelength Another example is OD measurement in bacterial cell cultures at 600 nm Measurements such as absorbance or concentration directly if an appropriate factor is applied as a function of time either in minutes or seconds can be made with your instrument Delay time defines at what time into the assay the spectrophotometer starts to collect data thereby accounting for a lag phase Time Interval defines the time between each absorbance measurement Duration is the total length of time including delay time if applicable for the assay The presentation of results can be either as actual absorbance or concentration at each defined time interval period RAW DATA
20. ent users of the UV range can benefit from considerable increase in deuterium lamp life by use of this utility SOUND Switch sound on or off when keyboard and Sipper if fitted buttons are pressed and at intervals in Rate modes RUN PRINT Prints what is currently shown on the display sending it to the printer every time RUN is pressed or when the display is updated as in Reaction Rate GLP PRINT OUT Print out a header at each calibration or re calibration with information to conform with GLP if a printer is connected setting GLP to OFF does not affect instrument calibration there is a 10 minute delay while the instrument warms up Pressing enter after calibration confirms that the instrument status is acceptable In addition when run is pressed the underlying readings and results are printed out RE CALIBRATE Causes instrument to re calibrate see GLP PRINT OUT LAMP CHANGE AT Set the cross over wavelength at which instrument changes from UV deuterium to visible tungsten lamp range is from 290 to 380 nm default is 325 nm Use only if there is critical detail at or around 325nm LOCK UNLOCK Lock instrument into its current operating mode by entering a four digit number When instrument is locked you can only cycle between Absorbance Locked Mode and Transmission on using MODE lt gt keys Unlock instrument from its current operating mode by entering the four digit number used to lock it
21. essor controlled instrument It works on the basis of light from either of the lamp sources being directed by a motorised mirror through the monochromator inlet slit This passes through one of several dependent on wavelength selected filters mounted on a filter quadrant the filtered light is then directed onto the holographic grating which produces light of the selected wavelength The light then leaves the monochromator via the exit slit and mirrors focus and direct the light into the sample compartment This passes through your cell containing the sample of interest and a defocussing lens to a solid state detector unit The resulting signal is then amplified and displayed Your spectrophotometer measures standard absorbance concentration and transmittance has stored parameters for DNA RNA and oligonucleotide quantification and purity checking as well as for protein contamination measurement in nucleic acid solutions measures absorbance ratios measures change of absorbance with time and can either print the numerical data or output the absorbance time plot to a chart recorder synchronised outputs a wavelength scan to a chart recorder asynchronous can be connected to a standard centronics parallel printer for output of results can be linked to a PC via its serial interface and used with the SWIFT range of software application programs operating in the Windows envi
22. ficient it should be set to zero a negative coefficient arises because there is no contribution to the protein concentration due to the absorbance at 260 nm The effects of background absorbance can be compensated for if required Reference Warburg and Christian Biochemisches Zeitung 310 384 1941 Abs Protein Conc Factor 1 x Abs id 1200 Abs p Factor 2 x Abs ww 2280 4320 background is optional Factors can be changed from defaults Set factor 2 0 00 for direct A280 UV protein measurement If BSA bovine serum albumin is an acceptable standard setting factor 1 1 115 will give linear results from 0 to 0 8 mg ml mode gt PROTEIN IMPURITY enter Background Y N background lt gt emer Factor1 1 550 Factor 1 enter Factor 2 0 760 Factor 2 enter 8 g Set Reference fun gt 0 510 C PRO 1 Protein impurity mg ml Issue 05 01 2000 English 15 ABS RATIO DNA RNA Oligo modes will give quantification results in ug ml directly if a 10mm pathlength cell and no dilution by using the conversion factors of 50 40 and 33 respectively To get results directly using a 5mm pathlength cell and or a solution which has had for example a 10 times dilution the factor must be changed To do this use Abs Ratio mode and set wavelengths accordingly If using 5mm pathlength cell for DNA quantification us
23. gth cell Oligonucleotides as a rule of thumb have a corresponding factor of 33ug ml although this does vary with base composition Use Factor concentration mode if you wish to use a different factor Concentration A o Factor Nucleic Acid Purity Checking Absorbance ratio can be used to establish the presence of impurities in a sample preparation relative to a pure sample The two wavelengths of interest to the Molecular Biologist are the absorbance maxima of the nucleic acid 260nm and the protein impurity 280nm A deviation from the expected absorbance ratio of the two wavelengths for the pure substance indicates the presence of impurity in the sample Absorbance ratio A A 2260 A280 The A 9 Ago absorbance ratio is known for the pure nucleotide 1 8 for pure DNA Di 0 for pure RNA enabling rapid assessment of quality Background correction A wavelength which is totally separate from these peaks 320nm is sometimes used to compensate for the effects of background absorbance due to for example turbidity or high absorbance buffer solution Concentration A Factor 1260 Ay 300 Abs ratio A A 2260 Pisa 2280 A 30 If your laboratory has not used background corrections before set this option to NO Reference Molecular Cloning Maniatis et al 12 English Issue 05 01 2000 Summary Nucleic Acid sample type Factor ug ml Az Aso ds DNA 50 1 8 RNA 40 2 0 Oligonucleotide 33
24. h on printer or deselect RUN PRINT and GLP PRINT OUT Issue 05 01 2000 Heated accessory Plug in and or press Temperature failed unplugged Contact service engineer Call Service E and advise diagnostic error code i Refer to Appendix Waiting for GLP Shen warming wp GLP Diagnostic Tests Detector seeing too lt Min A ae on WOE anes much light Ensure i baseplate plug is fitted Close lid properly Sample too concen gt Max A aa of range trated Something i blocking light path Detector did not see Replace the visible Vis mirror fail enough energy during lamp calibration Accessory Messages After installation of accessory press 4 cell changer 4 position cell changer 6 cell changer All 6 position cell changers All single cell holders Single Cell Holder except Electric Cell Electric Cell Holder Thermostat holder Peltier Cell Holder Issue 05 01 2000 English 27 ACCESSORIES All cell holders accommodate 10 mm pathlength cells as standard unless stated otherwise Refer to Essential Safety Notes on page 3 Multiple Cell HolderAccessones Install by removing existing cell holder replacing with the new one and turning the central fixing screw until it is finger tight followed by unction 4 position cell changer 8 position water heated cell changer 80 2106 01 80 2109 70 Accessory 4 cell changer
25. has warmed up at a constant ambient temperature and are typical of a production unit As part of our policy of continuous development we reserve the right to alter specifications without notice Wananty Your Supplier guarantees that the product supplied has been thoroughly tested to ensure that it meets its published specification The warranty included in the conditions of supply is valid for 12 months only if the product has been used according to the instructions supplied Your Supplier can accept no liability for loss or damage however caused arising from the faulty or incorrect use of this product Biochrom Ltd 22 Cambridge Science Park Milton Road Cambridge CB4 OFJ England Telephone 44 1223 423723 Fax 44 1223 420164 Issue 05 01 2000 English 43 44 English Issue 05 01 2000
26. ical surfaces of either lamp with your fingers use tissue if touched the area should be cleaned with isopropanol Ste I Issue 05 01 2000 English 35 To replace a lamp proceed as follows 1 2 3 4 5 6 7 8 9 36 Switch off the instrument remove sample from cell holder and disconnect the power supply cord Locate the lamp access cover on the left hand side of the instrument push down on the recess and pull the cover off Undo the fastening screw and remove the cover plate Slide the lamp plate out and unplug the cable connector If the tungsten lamp has failed the replacement should be inserted onto the plate pushing it all the way down into its holder If the deuterium lamp has failed insert the tungsten lamp onto the plate as above and then replace the whole assembly with the new one Reconnect the cable connector and slide the lamp plate in until it locates Replace the black covering plate by re tightening the fastening screw Replace the lamp access cover Reconnect the power supply cord and switch the instrument on Reset lamp life to zero and take an original energy reading as appropriate by Set up V RESET LAMPLIFE SAVE BASELINE Y N gt Enter This is needed for your GLP PRINT OUT English Issue 05 01 2000 Deuterium Lamp Wa nanty Criteria for lamp replacement are that it must be less that 1
27. ill drain onto the bench or table under the spectrophotometer or if preferred this drain hole can be connected to waste using suitable tubing Remove the cell holder and clean separately Use a soft dry cloth to mop out the sample compartment Replace the cell holder Reconnect the power cord and switch on the instrument 38 English Issue 05 01 2000 APPENDIX Good Laboratory Practice Good laboratory Practice GLP concerns being able to trace experimental results to an instrument an operator and the time the result was obtained so that a labora tory can prove that the instrument was functioning correctly or not Laboratory operator and internal instrument reference names can be entered on the spectrophotometer On calibration or re calibration the instrument checks the parameters and presents the status for the customer s acceptance prior to going into normal operation If GLP PRINT OUT is ON a print out of the results of these tests is then done The date time and operation are printed when parameters are printed GLP requires that the readings underlying any result shall be recorded and GLP PRINT OUT is ON performs this task automatically and prints the results GLP Diagnostic Tests The integrity of the instrument for GLP purposes is quantified from the calibration status of the instrument the age and energy of the lamps compared to their values when new the wavelength accuracy by co
28. ill show the wavelength used and the absorbance and cell position of the sample in the light path If run is pressed the sample absorbance is measured and the cell changer rotated sequentially to the next position the display therefore shows the wavelength used and the absorbance and cell position of the next sample since it is now in the light path The advantage of using run is that if a printer is connected and RUN PRINT in Setup selected then there is automatic print out of the absorbance details of the sample just measured Using with 2 samples Mode gt 360 0 000 A 1 wave Wavelength 546 4 etref 546 0 000 A 1 4c 546 1 200 A 1 Emple cell 2 546142A 2 Using with 2 samples 360 0 000 A 1 Wavelength 546 tref 546 0 000 A 1 546 1 200 A 1 546 1 412 A 2 546 0 000A 3 English enter enter Set A remove blank Absorbance of sample 1 cell position enter Absorbance of sample 2 enter Set enter remove blank Absorbance of sample 1 Un Absorbance of sample 2 cell position 3 empty Issue 05 01 2000 to set absorbance to 0 000 AU on a reference solution at all wavelengths in the mode selected During a standard operating procedure the user is prompted to insert a cell containing reference into cell holder by the message until the reference is inserted set reference
29. mparing to the 656nm deuterium line the values of a built in absorbance filter compared to when the instrument was manufactured or last serviced by an accredited engineer the bandwidth at 656nm the instrumental stray light The expected values are given in parentheses on the GLP print out after calibration and the range of acceptance is defined in the technical specification of the instrument In the unlikely event that the instrument fails calibration or goes out of specification a sequence of error messages will appear on the display the final message being GLP CALIBRATION FAIL These should be acknowledged sequentially by pressing enter and checking them with reference to the MESSAGES section of the user manual If a parallel printer is connected doing so will provide further information on the GLP print out The following should be checked is the cell compartment lid closed properly is a sample in the light beam if so remove it is the baseplate plug in place single cell accessory is the blanking plug at the front of the cell compartment in place Issue 05 01 2000 English 39 Pressing enter after the message GLP CALIBRATION FAIL appears on the display confirms that you have accepted the instrument status This status does not necessarily mean that the instrument cannot do the measurement required by the protocol for your experiment but having looked at the GLP print out you may wish to contact your
30. mplete manually See earlier if you wish to enter your details for printing out of power of lamp measured at calibration relative to when the lamp life was last set to zero 42 English Issue 05 01 2000 SPECIACATION Wavelength range Monochromator Spectral bandwidth Wavelength accuracy Wavelength reproducibility Light sources Detector Photometric range Photometric accuracy Photometric reproducibility Stability Stray light Analogue output Digital output Sample compartment size Dimensions Weight Power requirements Safety Standard EMC emissions EMC immunity Mains harmonies Susceptibility Standard Quality System 190 1100nm 1200 lines mmAberration corrected concave grating 3nm Inm 0 5nm tungsten halogen and deuterium lamps silicon photodiode 3 000 to 3 000A 0 01 to 99999 concentration units 0 1 to 200 T 0 5 or 0 003A to 3 000A whichever is the larger within 0 5 of absorbance value 0 001A per hour at 340nm tungsten lamp lt 0 025 T at 220nm using Nal and lt 0 025 T at 340nm using NaNO 100mV per 1 000A i 9 pin serial and Centronics parallel 210 x 140 x 80mm 500 x 360 x 190mm 13kg 90 265 V AC 10 50 60Hz 150VA EN61010 1 EN 50 081 1 Generic emissions part 1 EN 50 082 1 Generic immunity part 1 EN 61000 3 2 IEC801 Designed and manufactured in accordance with an ISO9001 approved quality system Specifications are measured after the instrument
31. nchronised Chart recorder cable 80 2105 95 is required Suggested chart recorder settings are 10mm second 200 mV full scale deflection output is 100 mV for 1 000 Abs unit recorder on zero suppress on zero set to internal The instrument will beep at every time interval if sound is ON To change this go to Set up See Instrument Utilities To abort an assay press STOP It is not possible to perform parallel kinetics or multi samples with this mode To do this use the SWIFT KIN software module which is available for use on PC LF L gt I g 18 English Issue 05 01 2000 RATE DELTA DATA display of change of absorbance with time KND RATE DELTA DATA Set Wave 600 SetX Time unit sec min Seconds or Minutes lt gt Delay 0 Delay Time 0 lt 999 Interval 1 Time Interval Is 99 po s00 Hon mc nig debs IfNO Factor 1 000 Factor Set reference YIN Set reference If Yes lt gt Gul 600 0 375 A KND 1 Gan 600 0010 A 0 03 18 updated every time interval The instrument will beep at every time interval if sound is ON To change this go to Set up To abort an assay press STOP Change in absorbance data can only be output to a parallel printer and not to a chart recorder gt RS Issue 05 01 2000 English 19 Scan An absorption spectrum can be obtained with your inst
32. nd the sipper It is essential that the baseplate plug supplied with single cell accessories is fitted to optimise air flow and prevent light ingress Symbolsused in Manual Please Note Display Panel lt gt Key Press Key Press Warning Abs Absorbance Wavelength Insert cell containing Insert cell containing reference into cell sample into cell holder holder Issue 05 01 2000 English Keyboard OO OOO OOO So ee Ignoring the numeric keypad the keyboard is designed to be operated from top to bottom and from left to right Basic absorbance measurements are very easy to make In addition a range of instrument utilities and instrument modes is accessi ble when the function or mode key is pressed these options are described later Select measurement mode required using lt I gt l Keys Press Select wavelength either from the numeric keypad or by using lt gt keys Press Set absorbance and transmittance readouts to 0 000 absorbance units and 100 T respectively on a reference solution BD VG OC lt Vv ar Vv NH Select sample position in cell changer either from the numeric keypad or by using lt keys Press Select options on the display Clears a numeric entry Start measurements when operating from within the measurement modes
33. numeric entry Issue 05 01 2000 English a myn A ig 41 GLP print out at instrument calibration GLP PRINT OUT on Example Laboratory Male uoegina dna Saree Instrument Ref Leve se anice Ultrospec 2000 UV Vis Spectrophotometer Serial no 3 63035 Software 4090 V2 0 19951115 Slave V1 7 Last serviced 19960219 Instrument state at calibration Calibrated 19960219 at 15 12 Calibration 2 point valid 190 1100nm Bandwidth at 656nm 3nm 2 6nm PASS Wavelength 656nm 656 0nm PASS Absorbance expected read at 220nm 2 414 2 417 z PASS at 340nm 1 986 1 1 992 e PASS at 500nm 1 488 1 489 3 PASS Stray light at220nm lt 0 025 T 0 0125T PASS UV Lamp 101 of original energy Vis lamp 100 of original energy Current instrument state Accessory six position cell changer There are no reference readings made by the cell changer UV lamp installed 19960219 at 10 03 total use 5 hours baseline in use dated 9960219 at 15 01 stored baseline date 9960219 at 15 01 Vis lamp installed 9960320 at 10 03 total use 41 hours baseline in use dated 9960219 at 15 02 stored baseline date 9960219 at 15 02 et j J The GLP and header print outs are obtained after instrument calibration and assumes a printer is connected and the GLP Print Out option is ON The instrument waits 10 minutes to allow the lamps to warm up properly These are printed out for you to co
34. ronment A range of accessories further enhances the capability of the instrument 6 English Issue 05 01 2000 MeasurementModes The mode key gives access to the 6 operational modes these are linked in a continuous loop After pressing mode use the arrow keys to move up down left right in the matrix shown below Each mode is described subsequently Select from the table below with lt I gt Xu ED gt Abaibance lt gt Concentaton FACTOR CONCENTRATION STANDARD CONC gt ada San RATE RAW DATA SCAN TO RECORDER RATE DELTA DATA Nucleic Acids DNA RNA OLIGO PROTEIN IMPURITY ABS RATIO Transmission lt IOAN to choose required measurement mode By pressing the mode button within a measurement mode the user can return to the previous step of operation The instrument can be switched off at any stage of operation A flashing cursor indicates an option which is currently selected Issue 05 01 2000 English Absorbance mode is the default after power on It is used to perform simple absorbance measurements on samples and it measures the amount of light that has been passed through a sample relative to a blank this can be air The procedure is to select the appropriate wavelength insert blank and set reference on it remove the blank and insert the sample s If sample is pressed and cell position 2 selected the display w
35. rument by output to chart recorder Ensure chart recorder is connected as scan data is not visible without this The Amersham Pharmacia Biotech REC 101 is recom mended SCAN TO RECORDER Start Wave 360 Start A End Wave 560 Finish A Set Reference Set ref n Scanning Chart position as scan is output Renter a OONN to recorder AFTER scan has 560 0 209 A SCN 1 finished Output to the Amersham Pharmacia Biotech REC 101 chart recorder is asynchronous the chart paper drive is controlled on off automatically Chart recorder cable 80 2105 95 is required Suggested chart recorder settings are 10mm second 200 mV full scale deflection output is 100 mV for 1 000 Abs unit recorder on zero suppress on zero set to internal If you require post run data manipulation routines the SWIFT SCAN software module is available for use on PC I te 20 English Issue 05 01 2000 Transmission In transmittance mode the instrument measures the amount of light at the specified wavelength that has passed through the sample and compares it with that which has passed through the reference This is displayed as a percentage The relationship between the concentration of the sample and its transmittance at any given wavelength is not linear and hence transmittance mode is rarely used experimentally except for samples having very high absorbances low transmitt
36. sequence dependent Notes If you wish to use other factors for example by incorporation of a dilution factor or other wavelengths for example if the peak maximum is at 257nm or a background wavelength at 350 nm use Absorbance Ratio mode ABS RATIO When using these modes with restricted aperture cells for example the 7ul ultramicrovolume cell ensure that they are correctly filled by holding up to the light This is to avoid the possibility of beam clipping and ensures that the results obtained are reproducible Background compensation at 320nm is used to correct for high absorbance buffer solution It is also helpful if using low absorbance solutions in ultramicrovolume cells If a printer is connected the results including absorbance values at 260 280 and 320 nm if selected are printed out automatically If a printer is not connected the absorbance values are displayed for a few seconds in turn before showing the concentration and ratio results DNA Mode gt DNA Background YIN background lt gt enter gt Set Reference Co 31 00 C DNA 1 DNA concentration ug ml ANNM rat 1 750 DNA 1 A260 A280 ratio Issue 05 01 2000 English 13
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