GeneQuant pro User Manual
Contents
1. C into holes and slot in mounting plate Refit two screws B and tighten carefully Refit connector A pushing downwards until the catch snaps shut 6 Refit the instrument top cover taking care not to trap the ribbon cable 7 Refit the seven screws in the base 8 Reconnect the power supply cord and switch the instrument on Issue 06 12 2003 GeneQuant pro English 30 SPECIFICATION Survey scan from 220 330 nm Monochromator Czerny Turner configuration with 1200 lines mm holographic grating Wavelength calibration Spectral bandwidth Wavelength accuracy Wavelength reproducibility Light source Detector 0 to 2 000A for 546 562 595 600 nm greater Photometric reproducibility Stray Light 9 pin serial via interface adapter lead Dimensions Weight Power requirements EEE Safety standard EMC emissions EMC immunity Susceptibility standard ISO 9001 approved quality system Specifications are measured 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 Issue 06 12 2003 GeneQuant pro English 312. Insert reference and press set ref Insert sample and press enter or Tm The melting temperature based upon the oligonucleotide concentration actually measured in the sample is displayed this is done by applying the nearest neighbour method to the entered base sequence and uses the salts molarity entered above Press stop to exit Example of output Base Sequence AGC AGC AGC AGC AGC Phosphorylated Counter Ion Buffer Molarity Primer Conc Calculated Tm No 260nm 1 0 123 Yes Na 0 100 1 000 57 0 degC Measured Tm 57 8 Issue 06 12 2003 GeneQuant pro English 19 Protein 595 Assay Refer to Techniques for further information Method No of standards Pathlength Units New standards Standard 1 Standard 2 Insert sample Select Bradford Biuret or BCA The selected method becomes the default Key in the number of standards range 3 27 Select 10 5 2 1 or 0 5 mm Select ug ml mg ml ug Select Yes or No appears only if no method has been entered If No last standards entered are used If Yes insert reference and press set ref Key in the concentration of the first standard range 0 0 1000 0 Insert Standard I and press enter The absorbance is measured and displayed together with the concentration of the standard Press enter Key in the concentration of the next standard range 0 0 1000 0 Insert Standard 2 and press enter
3. GeneQuant pro User Manual English Deutsch Fran ais Espa ol Italiano RUDE lochrom Biochrom Ltd Certificate No 890333 Declaration of Conformity This is to certify that the GeneQuant pro UV Vis Spectrophotometer Part number 80 2114 98 99 and 80 2115 04 05 Serial number 79000 onwards manufactured by Biochrom Ltd conform to the requirements of the following Directives 73 23 EEC amp 89 336 EEC Standards to which conformity is declared EN 61 010 1 2001 Safety requirements for electrical equipment for measurement control and laboratory use EN 61326 1998 Electrical equipment for measurement control and laboratory use EMC requirements Signed Dated 23 October 2002 a David Parr Managing Director Biochrom Ltd Postal address Telephone Telefax Biochrom Ltd 44 1223 423723 44 1223 420164 22 Cambridge Science Park Milton Road e mail enquiries biochrom co uk website http www biochrom co uk Cambridge CB4 OFJ England Registered in England No 974213 Registered Office 22 Cambridge Science Park Milton Road Cambridge CB4 4FJ England Issue 06 12 2003 GeneQuant pro English 1 CONTENTS Unpacking Positioning and Installation Essential Safety Notes Rear panel OPERATION Introduction Instrument Description Utility keys Reading keys TECHNIQUES DNA RNA and oligonucleotide characterisation Nucleic Acid Quantification NAQ Nucleic Acid Purity Checks Use of B
4. Issue 06 12 2003 GeneQuant pro English 11 Protein Determination Protein Determination at 595 546 and 562 nm The Bradford method depends on quantitating the binding of a dye Coomassie Brilliant Blue to an unknown protein and comparing this binding to that of different known concentrations of a standard protein at 595 nm this is usually BSA bovine serum albumin The Biuret method depends on reaction between Cupric ions and peptide bonds in an alkali solution resulting in the formation of a complex absorbing at 546 nm The BCA method also depends on reaction between Cupric ions and peptide bonds but in addition combines this reaction with the detection of Cuprous ions using bicinchoninic acid BCA giving an absorbance maximum at 562 nm The BCA process is less sensitive to the presence of detergents used to break down cell walls Detailed protocols are supplied with these assay kits and must be closely followed to ensure accurate results are obtained Due to the low lamp energy at 546 595 nm absorbances greater than 2 000 cannot be measured The use of plastic disposable cells is recommended To include the zero concentration standard include it in the number of standards to be entered and enter 0 00 for concentration use this when required to enter standard 1 If using duplicates enter the same concentration twice 2 duplicates of 3 different concentration equals 6 standards Set reference on water A linear regression analysi
5. print base seq Enables entry of a primer or oligonucleotide base sequence for calculation of Theoretical Absorbance Molecular Weight and conversion factor see Base Sequence Entry Tm Enables determination of Tm from Abs260 and display of calculated Tm for an entered base sequence see Tm stop Behaves as an escape key moving the user back to Instrument Ready set ref Reads reference values at appropriate wavelengths enter Enter accept option in set up and acts as reading key Issue 06 12 2003 GeneQuant pro English 7 Reading keys measures absorbances at calculates measure on 230 260 280 320 595 600 nm no results are calculated individual samples protein 595 assay protein 280 meas cell culture measures absorbances at calculates measure on measures absorbances at calculates measure on measures absorbances at calculates measure on measures absorbances at calculates measure on measures absorbances at calculates measure on measures optical density at calculates measure on 230 260 280 320 nm DNA concentration 260 280 and 260 230 ratio individual samples 230 260 280 320 nm RNA concentration 260 280 and 260 230 ratio individual samples 230 260 280 320 nm oligo primer concentration 260 280 ratio and 260 230 ratio individual samples 595 546 or 562 nm protein concentration individual samples relative to a calibration
6. 001 ug ul pmol ul ug ml x 1000 MW of oligo pmol phosphate nucleotide concentration ug ml 315 The molecular weight MW of a DNA oligonucleotide is calculated from MW g mole dA x 312 2 dC x 288 2 dG x 328 2 dT x 303 2 MW counter ion x length of oligo in bases for RNA oligonucleotide dT x 303 2 is replaced by dU x 298 2 The MW calculated using this equation must be adjusted for the contribution of the atoms at the 5 and 3 ends of the oligo Phosphorylated oligos add 17 2 x MW counter ion Non phosphorylated oligos subtract 61 MW counter ion The MW g mole of the most common oligo counter ions are Na sodium 23 0 K potassium 39 1 and TEA Triethylammonium 102 2 Issue 06 12 2003 GeneQuant pro English 9 Nucleic Acid Purity Checks The extraction of nucleic acids from cells is accompanied by protein and extensive purification is required to separate the protein impurity The 260 280 ratio gives an indication of purity it is only this however and not a definitive assessment Pure DNA and RNA preparations have expected ratios of gt 1 8 and gt 2 0 respectively deviations from this indicate the presence of impurity in the sample but care must be taken in interpretation of results The 260 nm reading is taken near the top of a broad peak in the absorbance spectrum for nucleic acids whereas the 280 nm reading is taken on a steep slope i e small changes i
7. The absorbance is measured and displayed together with the concentration of the standard Press enter Repeat procedure until all standards have been measured The standard curve details slope correlation coefficient and intercept are displayed Insert reference and press set ref Insert sample and press enter The absorbance is measured and displayed together with the concentration of the sample relative to the standard curve Repeat procedure until all samples have been measured Press stop to exit Issue 06 12 2003 GeneQuant pro English 20 Example of output protein 595 Date 22 May 2000 Name Method Bradford No of standards 6 Abs Conc 0 693 0 139 0 842 0 278 0 934 0 417 1 026 0 556 1 150 0 650 1 285 0 834 Slope 0 746 Corr Coeff E 0 996 Intercept I 0 593 No A Conc 1 0 520 0 162 Issue 06 12 2003 GeneQuant pro English 21 Protein 280 Measurement Refer to Techniques for further information Coeff 1 Key in the factor to be used for 280nm default is 1 550 range is 0 01 10 00 Coeff 2 Key in the factor to be used for 260nm default is 0 760 range is 0 00 10 00 Use 320nm Select yes or no Insert reference and press set ref Insert sample Insert sample and press enter or protein 280 The absorbances are measured and displayed together with the calculated concentration Press stop to exit Example of output Pro
8. specification if you prefer to do it in house instructions for carrying this out are provided with the filters e Observe all necessary precautions if dealing with hazardous samples or solvents Issue 06 12 2003 GeneQuant pro English 28 Cleaning and General Care of the Instrument External cleaning e Switch off the instrument and disconnect the power cord e Use a soft damp cloth to clean all external surfaces e A mild liquid detergent may be used to remove stubborn marks Sample compartment area spillage e Switch off the instrument and disconnect the power cord e The sample area is manufactured from a chemical resistant material Strong concentrations of sample however may affect the surface and spillages should be dealt with immediately e A small drain hole in the sample area allows excess liquid to drain away onto the bench or table from under the instrument e Use a soft dry cloth to mop out the sample area e Reconnect the power supply cord and switch on the instrument Fuse Replacement e 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 do not touch the power supply socket prongs e Slide the fuse holder open by pulling at the notch e Place fuses 1 6AT 5mm x 20mm FST into the fuse holder and slide back into positio
9. to 40 C e Maximum relative humidity of 80 up to 31 C decreasing linearly to 50 at 40 C The instrument must be placed on a hard flat bench or table that can take its weight 4 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 5 cm 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 Switch on the instrument at the rear panel the instrument initialises for a few seconds and then goes through a calibration procedure for a minute or so until the display shows Instrument Ready together with information relating to serial number software version date and sample number 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 Issue 06 12 2003 GeneQuant pro English 4 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 is yellow symbol and outline ar
10. 100 2500 gt 3 ul capillary 0 5 80 2104 66 spare capillaries 100 80 2104 67 assuming dsDNA with Abs 260 1 0 for 50 ug ml ng ul in a 10 mm pathlength cell this is supplied with a micro sample viewer accessory k note that dilution may not be required for a miniprep PCR amplification where typical concentrations are between 50 and 200 ng u1 e The 7 ul ultramicrovolume cell has a minimum detection limit of 2 ug ml dsDNA assuming an absorbance of 0 020 this equates to 14 ng dsDNA if 7 ul of solution is used This very low level is not sufficient for reproducible results Use of background correction is recommended e The capillary cell has a minimum detection limit of 20 ug ml dsDNA assuming an absorbance of 0 020 this equates to 60ng dsDNA if 3 ul of solution is used This very low level is not sufficient for reproducible results Use of background correction is recommended e The use of standard 1 mm and 5 mm pathlength cells is not recommended due to the requirement of a packing piece not required for the ultra microvolume and capillary cell described above Issue 06 12 2003 GeneQuant pro English 25 Comparison of instrument with other spectrophotometers e You must compare like with like using the same sample with the same cell and obtain absorbance measurements on both at the same wavelengths e GeneQuant pro has a bandwidth of 5 nm and this should be compared with a spectrophotometer having the same bandwi
11. Autoprint is on results for reading keys only will be output automatically use of the print key is disabled Issue 06 12 2003 GeneQuant pro English 15 DNA RNA and oligo Refer to Techniques for further information Pathlength Select 10 5 2 1 or 0 5mm Units Select required concentration units from ug ml ng ul ug u pmol ul pmol phosphate pmol ul is only valid for oligonucleotides and requires base sequence entry in order to be calculated Use 320nm Select yes or no Dilution Factor Key in the dilution factor for the concentration calculation range 1 1000 Insert reference and press set ref Insert sample Insert sample and press enter or DNA RNA as appropriate The absorbances are measured and displayed together with the 260 230 and 260 280 ratios and the concentration If the sample is too dilute to give reproducible results the concentration is displayed as invalid Press stop to exit oligo only Default Factor Default factor of 33 ug ml This can be changed for example to 37 for ssDNA if required Calc Factor Display shows the conversion factor ug ml if base sequence has been entered Use Default Select no if calculated factor is to be used if yes default factor is used Insert reference and press set ref Insert sample Insert sample and press enter or oligo The absorbances are measured and displayed together with the 260 230 and 260 280 ratios and the con
12. Select appropriate month Year Key in year Sample No Key in required number increments automatically default is 1 Output Mode Select output to Printer standard Centronics parallel lead required Serial a special serial lead is required for connection to PC or None AutoPrint Automatic output of reading key results to Printer or PC print key is disabled Select On or Off Survey Scan Select On Off or Print relevant to Nucleic Acid modes only Language Select English German French Spanish or Italian Use Date Select On or Off off means that date will not be printed Key Click Select On or Off Sample measurement e Press reading key to access the mode then set ref key to set reference e Measurements are taken at all relevant wavelengths and displayed as 0 000 values A Starting Lamp message is displayed prior to measurement e Press reading key again or enter in order to make readings e Measurements are taken at all relevant wavelengths and actual absorbance values are displayed together with the results of calculations A Starting Lamp message is displayed prior to measurement e Sample number is always displayed reset using set up e After measurement press print to obtain a print out comprising header selected set up options absorbance readings as appropriate and the various calculated results as appropriate Issue 06 12 2003 GeneQuant pro English 14 e If
13. ackground Correction Protein Determination Protein Determination at 595 546 and 562 nm Protein Determination at 280 nm Bacterial Cell Culture Measurement SET UP AND SAMPLE MEASUREMENT Set up Sample measurement DNA RNA and oligo Base Sequence Tm Protein 595 Assay Protein 280 Measurement Bacterial Cell Culture MESSAGES OUTPUT OF RESULTS Use with parallel printer Use with PC CELLS Selecting the appropriate cell Comparison of instrament with other spectrophotometers Filling cells Taking measurements Cleaning cells Reproducibility using reduced aperture ultramicrovolume and capillary cells w D MAS AO ONN AA L UAU K eN UJ N LU L HM NNN N 2 O NO AD RA N W NNN N FS L2 L2 LS rs WK NN O O O U Nn Issue 06 12 2003 GeneQuant pro English 2 ACCESSORIES MAINTENANCE After Sales Support Performance Verification Check Cal Check Utility Key Cleaning and General Care of the Instrument Fuse Replacement Deuterium Lamp Warranty Deuterium Lamp Replacement SPECIFICATION Issue 06 12 2003 GeneQuant pro English 27 28 28 28 29 29 29 30 31 Unpacking Positioning and Installation Inspect the instrument for any signs of damage caused in transit If any damage is discovered inform your supplier immediately Ensure your proposed installation site conforms to the environmental conditions for safe operation e Indoor use only e Temperature 10 C
14. apillary cells e Ensure cell pathlength is set to 0 5 mm e Place capillary cell holder in sample compartment e Insert capillary Issue 06 12 2003 GeneQuant pro English 26 Cleaning cells e After use cells should be cleaned with a dilute alkali e g 0 1 M NaOH and a dilute acid e g 0 1 M HCl wash followed by rinsing several times with distilled water More rigorous cleaning after difficult samples should be performed with a suitable liquid laboratory glassware detergent e g Decon following the manufacturer s instructions e The capillary cell can be dismantled for cleaning and removing a broken capillary by unscrewing the two screws on each side using the tool provided Reproducibility using reduced aperture ultramicrovolume and capillary cells e Check for correct operation range and sample integrity by inspection of all 4 wavelength readings for nucleic acid measurements e Ensure that set reference is renewed periodically preferably every 10 consecutive readings e Ensure background correction at 320 nm is on e Ensure sample dilution is such that sample Abs 260 is greater than 0 100 e Ensure cell is scrupulously clean ACCESSORIES Deuterium Lamp 80 2109 86 Printer Stand 80 2109 96 Seiko DPU 414 printer enquire Parallel interface cable Centronics 80 2071 87 Interface adapter for serial connection 80 2109 02 includes spreadsheet interface software Spare GeneQuant pro User Manual multi language 80 2110 89 G
15. centration Press stop to exit Survey Scan On e Survey scan is over the range 220 330 nm and takes about 10 seconds Conc 495 15 ugf ul 230 0 8804 260 1 9034 200 1 2824 E02 5 2 163 O lt 1 484 Insert sample e Lines on the graph indicate 230 260 280 and 320 nm e After the scan is complete results are displayed use select to view the graph e Press set up to change absorbance scaling of graph use select and enter to choose between 0 1 0 2 0 5 1 0 2 0 2 5 and Autoscale Issue 06 12 2003 GeneQuant pro English 16 Examples of output DNA determination Date 22 May 2000 Name eere 590939464 Factor 50 0 Units ug ml Pathlength 10mm Dilution Factor 1 Use 320 nm No No 230nm 260nm 280nm 320nm 260 280 260 230 Conc 1 0 230 0 471 0 255 1 850 2 045 23 5 Oligonucleotide determination Date 22 May 2000 WANE cd dee owen cwsananends Dflt Factor i 33 0 Units I ue ml Pathlength 10mm Dilution 1 Use 320 nm No No 230nm 260nm 280nm 320nm 260 280 260 230 Conc 1 0 168 0 325 0 183 1 780 1 934 10 7 Issue 06 12 2003 GeneQuant pro English Base Sequence Refer to Techniques for further information Select DNA or RNA Phosphorylated Select Yes or No Counter Ion Select Na sodium K potassium TEA triethylammonium or other counter ion Enter Molecular Weight of the counter ion being used Press enter to initiate base sequence entry
16. curve of known standards 260 280 320 nm protein concentration individual samples 600 nm corrected OD individual samples GeneQuant pro English 8 Issue 06 12 2003 TECHNIQUES DNA RNA and oligonucleotide characterisation Nucleic Acid Quantification NAQ Nucleic acids can be quantified at 260 nm because it is well established that a solution of DNA or RNA with an optical density of 1 0 has a concentration of 50 or 40 ug ml respectively in a 10 mm pathlength cell Oligonucleotides as a rule of thumb have a corresponding factor of 33 ug ml although this does vary with base composition this can be calculated if the base sequence is known see Base Sequence in Set up and below Concentration Abs260 Factor The instrument uses factors 50 40 and 33 as defaults for DNA RNA and oligonucleotides respectively and compensates for dilution and use of cells which do not have 10 mm pathlength dilution factor and cell pathlength are entered via set up If using the ultramicrovolume or capillary reduced aperture cells ensure they are correctly filled by carefully following the instructions supplied with the cell see Cells and that the correct pathlength has been entered Default units are ug ml but units of ng ul ug ul pmol ul and pmol phosphate can be selected via set up the pmol ul unit is useful for sequencing and PCR calculations involving primers The following conversions are used 1 ug ml 1 ng ul 0
17. d using calibration curves A calibration curve can be determined by comparing measured OD to expected OD Expected OD is determined by counting cell number using an alternative technique for example microscope slide method and converting to OD using the rule of thumb that 1 OD 600 8 x 10 cells ml for E Coli GeneQuant pro has much smaller optics than most conventional spectrophotometers and more scattered light is transmitted through to the detector resulting in lower than expected OD 600 values Results obtained by comparing measured OD 600 with expected OD 600 see above indicate that a correction factor of 2 0 is required to make the data comparable to larger instruments this factor is included as a default value in set up The use of 10mm pathlength disposable cells is recommended for optical density measurements of cell culture solutions to prevent the suspension settling too quickly and giving an OD that changes with time glycerol should be added to the sample Issue 06 12 2003 GeneQuant pro English 13 SET UP AND SAMPLE MEASUREMENT Set up e For instrument set up press set up Press the appropriate reading key followed by the set up key to access relevant options there is no set up for absorbance e Press select and enter to work through the various options e Press stop at any time to return to Instrument Ready status or to go back a level Day Key in date adjust daily Month
18. dth since this parameter affects absorbance values Filling cells Ensure cells are always clean and dry before use and between samples Standard 10 mm pathlength cells e fill so that the level of the liquid measures at least 20 mm from the cell bottom Microvolume cell 80 2103 69 e fil so that the liquid meniscus cannot intercept the light beam we recommend at least 70 ul is used Ultramicrovolume cell 80 2103 68 e fill so that the liquid meniscus cannot intercept the light beam we recommend at least 7 10 ul is introduced into the pipette fitted with an appropriate tip e insert tip into one of the conical openings of the cell and ensure the tip is pushed in firmly deliver the sample slowly into the cell while tilting it with the other conical opening upwards to ensure all air is expelled e place cell in micro sample viewer and hold up to the light when viewed through the eye piece the 3 X magnification lens enables confirmation that there are no bubbles or other impediments in the light path e ensure the faces of the cell are wiped clean Capillary cell 80 2104 66 and capillaries 80 2104 67 e dipping the capillary into the solution will normally introduce enough liquid for measurement e liquid can be prevented from leaking out using the Cristaseal supplied Taking measurements e Ensure cell pathlength is set to 10 mm or 5mm for the ultramicrovolume cell 80 2103 68 ensure cell orientation is always the same For c
19. e black Rear panel 1 2 3 1 Multi purpose interface socket 2 Power supply switch 1 on 0 off 3 Power supply socket Issue 06 12 2003 GeneQuant pro English 5 OPERATION Introduction Your GeneQuant pro RNA DNA Calculator is a unique yet simple to use spectrophotometer designed specifically for molecular biologists working in the life sciences including biotechnology and drug discovery research It has a pulsed deuterium borosilicate glass lamp for long life It measures absorbance at 230 260 280 320 546 562 595 and 600 nm and various relevant parameters are calculated automatically and displayed at the touch of a button The instrument is ideal for many routine molecular biologist activities it can determine the concentration in ug ml ng ul ug ul pmol ul or pmol phosphate and purity of nucleic acids DNA RNA and oligonucleotides after PCR amplification for hybridisation studies and for quantitating minipreps The wavelength at 260 nm is used to calculate quantification and together with those at 230 and 280 nm for purity checking calculations via 260 230 and 260 280 ratios The wavelength at 320 nm is used for background compensation A variety of cells including UV transparent disposable cells is now available for convenient sampling depending on sample concentration dilution factor and available sample volume A nucleic acid survey scan facility is available so that the spectrum of samples ca
20. eneQuant calibration check filter set 80 2109 88 Spare dust cover 80 2109 13 Basic UV Visible Spectrophotometry Booklet 80 2108 63 Issue 06 12 2003 GeneQuant pro English 27 MAINTENANCE After Sales Support We supply support agreements that help you to fulfil the demands of regulatory guidelines concerning GLP GMT e Calibration certification using filters traceable to international standards e Certificated engineers and calibrated test equipment e Approved to ISO 9001 standard Choice of agreement apart from break down coverage can include e Preventative maintenance e Certification Performance Verification Check Cal Check Utility Key e Verification of instrument performance is important for Good Laboratory Practise GLP purposes GLP requires that results obtained in an experiment can be traced back to an instrument and that the instrument can be proved to be working correctly e Using a calibration check filter set the instrument can be checked for absorbance accuracy at 230 260 280 320 595 and 600nm wavelength accuracy at 260 and 280nm and stray light at 260nm e An accredited service engineer with your supplier should have a set of filters secondary standards traceable to NIST and be able to verify the instrument performance a certificate for record keeping will be provided A calibration check filter set part number 80 2109 88 is available for periodic testing of the instrument to ensure that it is working to
21. equations solving these provides the two coefficients In cases where Factor 2 is found to be negative it should be set to zero since it means there is no contribution to the protein concentration due to absorbance at 260 nm Set Factor 2 0 00 for direct A280 UV protein measurement Factor 1 is based on the extinction coefficient of the protein If BSA bovine serum albumin is an acceptable standard setting Factor 1 1 115 will give linear results from O to 0 8 mg ml protein Protein mg ml 1 115 Abs 280 Rapid measurements such as this at Abs 280 are particularly useful after isolation of proteins and peptides from mixtures using spin and HiTrap columns by centrifuge and gravity respectively Bacterial Cell Culture Measurement Bacterial cell cultures are routinely grown to an OD 600 of approx 0 4 prior to induction or harvesting A linear relationship exists between cell number density and OD 600 up to approx 0 600 OD It is important to note that for turbid samples such as cell cultures the light intensity measured is the proportion of the scattered light reaching the detector and not the result of molecular absorption The amount of scatter is affected by the optics of the system distance between the cell holder and instrument exit slit geometry of this slit and the monochromator optics Different spectrophotometer types therefore give different responses for the same turbid sample to compare results they must be normalise
22. face adapter lead 80 2109 02 is required ensure output to serial is on in Set up The ASCII stream is output at 19 200 Baud via the 25 way D connector on the rear panel and can be picked up by a PC with Windows 3 1 installed Use the Hyperterminal emulator in Accessories to pick this up settings are Handshake None 19 200 Baud 1 stop bit 8 data bits 0 parity Comm port depends on which port the lead is connected to Output is automatic if the interface lead is connected to the instrument Issue 06 12 2003 GeneQuant pro English 24 CELLS Selecting the appropriate cell The instrument measures absorbance in the range 0 005 to 3 000 using a 10 mm pathlength cell in general spectrophotometers are at their most accurate in the absorbance range 0 100 to 1 000 The cell should be chosen based on sample concentration dilution factor and available sample volume Readings lt 0 05 approach the detection level of the instrument which decreases reproducibility ideally such levels should be avoided Use the table below as a guide to selecting the correct cell GeneQuant pro has the standard optical height of 15 mm cells of 8 5 mm optical height require the use of an adapter Concentration Available Suggested cell type Pathlength Part number range after dilution sample mm ng ul volume ul 5 125 gt 2000 standard 10 80 2002 58 gt 500 semi micro 10 80 2002 77 gt 70 ul microvolume 10 80 2103 69 10 250 80 2103 68
23. facility for oligonucleotide or primer press A C G T U keys as appropriate minimum 10 maximum 64 mer Press the delete to remove the last base entered and cancel to remove a base sequence press select to change from yes or no Press select to display the parameters calculated from the base sequence calculations Theor Abs Display shows theoretical absorbance in units of AU umole Calc MW Display shows molecular weight of entered base sequence and counter ions including corrections for phosphorylation this value is used in the calculation of concentration in pmol ul and the conversion factor Calc Factor Display shows the conversion factor ug ml of entered base sequence calculated from MW divided by Theor Abs Example of output base sequence Date 22 May 2000 AGC AGC AGC AGC AGC Base type DNA Phosphorylated Counter Ion Theor Abs Calc MW Calc Factor Issue 06 12 2003 GeneQuant pro English 18 Tm Refer to Techniques for further information a minimum base sequence of 10 mer needs to have been entered Primer Conc Buffer Molarity Calculated Tm Insert sample Key in concentration of primer range 1 0 100 0 pmol ul Key in molarity of salts in hybridisation solution range 0 1 10 0 Display shows Tm calculated using the nearest neighbour method applied to the entered base sequence and the primer concentration and the salts molarity entered above
24. is a 128 x 64 pixel back lit graphics liquid crystal and the keypad has 28 keys organised into 3 areas numeric keypad utility keys and reading keys including set up see inner front cover for diagram After switch on initialisation and the calibration process the instrument is ready for use Instrument Ready Note the following points e it may be necessary to set up the instrument for your measurement particularly for nucleic acid quantification where low volume and reduced aperture cells are used and for doing a Bradford protein determination e set reference is always required at the start of each measurement type insert reference and sample cells so that the light path direction is in the back to front axis of the instrument e press the appropriate reading or enter key after the sample has been inserted length of measurement time is dependent on the number of wavelengths being read all wavelengths are read in absorbance mode over a period of approx 12 seconds Utility keys set up Enables configuration for the various instrument functions see Set up Pressing set up after the appropriate reading key will activate the set up process for that mode with the first option being highlighted cal check Enables instrument to be tested for absorbance accuracy wavelength accuracy and stray light with special filters see Performance Verification Check results for base sequence and nucleic acid modes if scan option is on
25. lected via set up Issue 06 12 2003 GeneQuant pro English 10 Use of Background Correction e Background correction at a wavelength totally separate from the nucleic acid and protein peaks at 260 and 280 nm respectively is sometimes used to compensate for the effects of background absorbance The wavelength used is 320 nm and it can allow for the effects of turbidity high absorbance buffer solution and the use of reduced aperture cells The instrument can use background correction its use is selected via set up e If it is used different results will be obtained to when it is not used because Abs320 is subtracted from Abs260 and Abs280 prior to use in equations Concentration Abs 260 Abs 320 Factor Abs ratio Abs 260 Abs 320 Abs 280 Abs 320 Abs ratio Abs 260 Abs 320 Abs 230 Abs 320 e If your laboratory has not used background correction before set this option to NO e The use of background correction can remove variability due to handling effects if the capillary cell or ultramicrovolume cell is being used see Cells Spectral scan of nucleic acid obtained using SWIFT software Abs PURE NUCLEIC ACID POLY dAdT MWave 260 0 Abs 0 700 Wave 280 0 Abs 0 383 200 0 225 0 250 0 275 0 300 0 325 0 350 0 Wavelength Note e absorbance maximum near 260 nm and absorbance minimum near 230 nm e flat peak near 260 nm and steep slope at 280 nm e very little absorbance at 320 nm
26. n e Reconnect the power supply cord and switch on the instrument e Fuses are not normally consumed in an instrument s lifetime If they blow repeatedly contact your supplier Deuterium Lamp Warranty The criterion for lamp replacement under warranty is that it must be less than 36 months old Issue 06 12 2003 GeneQuant pro English 29 Deuterium Lamp Replacement Replacement lamps are available from your supplier using the part number 80 2109 86 deuterium lamp assembly The design of the lamp area is such that users are able to change their own lamps No lamp alignment is required as the lamps are pre aligned at manufacture The lamp becomes very hot in use Ensure it is cool before changing it Do not touch the optical surface of the lamp with your fingers use tissue if touched the area should be cleaned with iso propanol To replace a lamp proceed as follows 1 Switch off the instrument and disconnect the power supply cord 2 Release the instrument cover by unscrewing the seven screws in the base 3 Carefully lift top cover upwards tilt and place on the right side of the instrument taking care not to damage the ribbon cables and not to touch any of the circuitry in particular the heat sink 4 Depress catch A and lift connector away from circuit board Remove two screws B Lift deuterium lamp and bracket assembly away from mounting plate 5 Place new deuterium lamp assembly into position locating pins
27. n be viewed it can calculate annealing temperatures for primers prior to PCR upon entry of the oligonucleotide sequence and concentration together with the total buffer molarity this is done using the nearest neighbour thermodynamic data for each base in the nucleotide chain in relation to its neighbour Breslauer et al Proc Natl Acad Sci USA 83 3746 1986 it can be used for protein determination using the Bradford Biuret and BCA protein assays at 595 546 and 562 nm respectively samples are measured against a stored standard curve and the linear regression results of the straight line are printed out it can measure OD 600 for bacterial cell culture solutions in order to optimise induction and harvest times To facilitate electronic project archiving of experimental results all data can be downloaded directly to Excel using the appropriate interface adapter lead Results can also be printed to a standard Centronics parallel printer A calibration check filter set is available for periodic testing of the instrument for GLP purposes wavelength accuracy absorbance accuracy and stray light results of tests and status of instrument can only be viewed by downloading to PC or printing as an aid to record keeping The user friendly measurement system offers ease of use and ensures a long lamp lifetime minimising the cost of ownership of the product Issue 06 12 2003 GeneQuant pro English 6 Instrument Description The display
28. n wavelength cause large changes in absorbance Consequently small variations in wavelength at 280 nm will have a greater effect on the 260 280 ratio than will variations at 260 nm Thus different instruments of the same and different types may give slightly different ratios due to variations in wavelength accuracy but each instrument will give consistent results within itself Concentration will also affect 260 280 readings If a solution is too dilute the readings will be at the instrument s detection limit and results may vary because there is less distinction of the 260 peak and 280 slope from the background absorbance This is one reason why absorbances should be gt 0 05 for accurate measurements An elevated absorbance at 230 nm can indicate the presence of impurities as well 230 nm is near the absorbance maximum of peptide bonds and also indicates buffer contamination since Tris EDTA and other buffer salts absorb at this wavelength When measuring RNA samples the 260 230 ratio should be gt 2 0 a ratio lower than this is generally indicative of contamination with guanidinium thiocyanate a reagent commonly used in RNA purification and which absorbs over the 230 260 nm range The nucleic acid survey scan facility is particularly useful for RNA samples The instrument can display 260 280 and 260 230 ratios and compensates for dilution and use of cells that do not have 10 mm pathlength dilution factor and cell pathlength are se
29. ot Fan has failed or air grill is blocked Filter error Filter wheel has not aligned properly Grating error Grating has not aligned properly Issue 06 12 2003 GeneQuant pro English 23 OUTPUT OF RESULTS Use with parallel printer e We recommend the use of the Seiko DPU 414 thermal printer however any Centronics parallel printer can be used together with the appropriate cable If using a thermal printer ensure it is set up to print out in condensed printing mode 80 characters so that there is no wrap round refer to printer user manual for the Seiko DPU 414 this is in the DIP SW settings section Ensure output to printer is on in the Set up e Output is alphanumeric only and automatic when the Print key is pressed and a printer is connected and switched on Umlauts and accents are not printed out with letters if the instrument is set up to be in German French Italian or Spanish e The Survey Scan can be printed using the Seiko DPU 414 printer only ensure Survey Scan in set up is set to Print as On enables the facility on the instrument display only Use with PC NOTE A standard serial interface will not work 1 Download to Spreadsheet The serial interface adapter lead 80 2109 02 is required it is also supplied with the Spreadsheet Interface Software for direct download to Excel This macro is supplied on a floppy disc together with instructions for installation and use 2 Use with Hyperterminal The serial inter
30. s of the calibration standard data points is calculated the result together with the correlation coefficient is printed out A correlation coefficient of between 0 95 and 1 00 indicates a good straight line Protein Determination at 280 nm Protein can be determined in the near UV at 280 nm due to absorption by tyrosine tryptophan and phenylalanine amino acids Abs 280 varies greatly for different proteins due to their amino acid content and consequently the specific absorption value for a particular protein must be determined The presence of nucleic acid in the protein solution can have a significant effect due to strong nucleotide absorbance at 280 nm To compensate for this by measuring Abs 260 the equation of Christian and Warburg for the protein crystalline yeast enolase Biochemische Zeitung 310 384 1941 can be applied Protein mg ml 1 55 Abs 280 0 76 Abs 260 or Protein conc Factor 1 Abs 280 Factor 2 Abs 260 This equation can be applied to other proteins if the corresponding factors are known The instrument can determine protein concentration at 280 nm and Issue 06 12 2003 GeneQuant pro English 12 uses the above equation as default the factors can be changed and the use of background correction at 320 nm is optional via set up To customise the equation for a particular protein the absorbances at 260 and 280 nm should be determined at known protein concentrations to generate simple simultaneous
31. tein 280nm Measurement Date 22 May 2000 0 000 Use 320nm No No 260nm 280mn 320nm Conc 1 0 123 0 275 Bacterial Cell Culture Refer to Techniques for further information Factor Key in the correction factor you have derived from calibration plot to make results directly comparable with other spectrophotometers used in your lab default is 2 0 and is typical Insert reference and press set ref Insert sample Insert sample and press enter or cell culture The OD at 600 nm is measured and displayed together with the corrected result Press stop to exit Issue 06 12 2003 GeneQuant pro English 22 MESSAGES The following messages may appear during use of the instrument gt 2 000 Protein 595 and Cell Culture readings only valid up to 2 000 A Invalid Curve Standards have not been inserted in corrected order or absorbance values do not increase sequentially Solution too dilute For reproducible results a more concentrated sample or a longer pathlength cell should be used Too much light Sample compartment lid not closed properly Ref 1 error Check sample compartment area for blockage Ref 2 error Check sample compartment area for blockage Lamp failure Lamp failed to strike switch off and try again Lamp may need replacing if symptom persists Contact your supplier if the messages below or any other obvious fault messages appear and cannot be cleared by re initialising the instrument Lamp too h
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