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NanoPhotometerTM P-Class User Manual P 300 / P 330 / P

1. I 0 0 2 0 4 0 6 0 8 1 0 Ao An dA Slope Us R 0 001 0 006 0 005 0 099 0 005 0 1957 Sample 1 Time 00 00 10 Abs 0 226 Menu select using key pad numbers Sb Parameters Data Transfer Print Oats Set tO At Cursor Set tn At Cursor o o C3 Slope o o o Sample Number Save Method Output Options Version 1 0 Results Screen Step 13 Insert the reference sample and press Blank key Step 14 Insert the sample and press Sample to start the run Time min is displayed at the bottom of the screen and absorbance data are plotted on the graph as testing proceeds The table below the graph gives absorbance values at Ao start of calculation An finish of calculation dA change in absorbance slope regression parameter R2 of the calculated slope and the result calculated from the selected parameter Step 15 Use the left and right arrows to move the cursor and display the time and absorbance value at measured data points Use the up and down arrows to zoom in or out Step 16 Press Menu Options to display available options which are described below Step 17 Press Escape O and confirm with Yes O to return to the Functions folder Query needs confirmation to avoid unintended escaping the application Menu select using key pad numbers 1 Return to parameter screen 2 Transfer the results via selected Ou
2. Background 03 2 100 optional 0 3 2 l 1 100 250 optional 0 3 2 l 1 250 Step 5 Select subsequent parameters and specifications as described under 4 Nanovolume Applications and Cuvette Applications After the selections are confirmed the results screen displays in top left corner the chosen Lid and the required sample volume Version 1 0 Page 10 68 Miser NanoPhotometer P Class User Manual Important Information If the absorbance value of the sample is not in the linear range the following Warning messages will appear and Instruction will be displayed in the top left corner of the result screen Concentration too low Concentration too high Please change to lid 10 and press sample No changes Do you want to change the lid factor automatic change of lid factor lid 5 to lid 10 in the software for calculation Concentration too high Please change to lid 50 and press sample No changes Do you want to change the lid factor automatic change of lid factor lid 10 to lid 50 in the software for calculation Lid 50 Concentration too low Please change to lid 10 apply a minimum of tul of No changes Do you want to change the lid factor sample and press sample automatic change of lid factor lid 50 to lid10 in the software for calculation Concentration too high Dilute sample or change to lid 100 the software for calculation Lid 100 Concentration too low Please change to lid 50
3. Lo aad 04 O16 O88 LO Le LY LE 2 Back Bradford Calibration 0 200 0 058 4 se 0 400 0 135 A BE H 0 600 0 2304 0 800 0 462 4 D4 ae 1 000 0 592 4 Fa 1 400 0 7194 Ta oe 1 600 C842 A ra ns 04 O86 DB LO Le L4 LG ok Calibration Screen replicates on Bradford Calibration Replicates 4 Replicates 1 D 718A D E ee 2 0 7188 va 3 07184 H eee Kai D 718A A mE Hd TE 04 06 DB LO Le Loy Version 1 0 Calibration Screen replicates off Step 14 This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 15 Insert the standard use C to clear previously stored results before measuring Press Next Ng to measure the standard and store the result Step 16 Repeat for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 17 When all standards are measured press ox to accept the calibration and go to the Results screen See below OR press Back Bio cancel selections and return to the Standards screen Calibration Screen replicates on Step 18 This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key Th
4. Open Output Options settings possibility to change the Output Options settings within the method as described in 7 3 Output Options Printer Exit Menu by pressing Escape Q OR wait Ch Output Data Only Seah e Sample Number Save Method Output Options OO8 Version 1 0 Page 8 68 Miser NanoPhotometer P Class User Manual 3 THE NANOPHOTOMETER P CLASS SUBMICROLITER CELL With its innovative optical pathway the cell is designed for optinum measurement results with submicroliter samples ranging from 0 3 ul up to 5 ul of undiluted sample Due to a pathlength of 0 04 mm 0 1 mm 0 2 mm 1 mm and 2 mm the cell is offering an automatic dilution of 1 250 1 100 1 50 1 10 and 1 5 in comparison to a standard cuvette measurement Because the measurements are processed with undiluted samples the reproducibility of the results is extremely high If desired samples can be retrieved after the measurement for further processing The NanoPhotometer P Class Submicroliter Cell can be used for all UV Vis analysis utilizing the wavelength range of 190 nm to 1 100 nm The NanoPhotometer P Class Submicroliter Cell is delivered for version P 300 with one lid with a pathlength of 0 2 mm Lid 50 for version P 330 with two lids pathlength 0 2 mm Lid 50 and 1 mm Lid 10 and for version P 360 with three lids pathlength 0 04 mm Lid 250 0 2 mm Lid 50 and 0 1 mm Lid 10 Lid 5 2 mm pathlength Lid 100 0 1 mm pathlength
5. Restore folder restores an individual folder from the SD Memory Card to the instrument Backup all folders generates a copy of all folders on the SD Memory Card Restore all folders restores all folders from the SD Memory Card to the instrument 3 Select the method to be saved using the left and right arrows 4 Press OK to save the method OR Cancel QO to return to the User Methods folder Version 1 0 Page 54 68 Miser NanoPhotometer P Class User Manual Methods Methods 1 0A Single Wavelength Gb Delete Method i E Lock Method Unlock Method Delete Method 1 Select the method to be deleted using the key pad numbers 2 Select Menu Options and press 1 Delete Method 3 Press OK to delete the method OR Cancel to return to User Methods folder Version 1 0 Page 55 68 Miser 7 UTILITIES NanoPhotometer P Class User Manual Survey of the available Utilities Utilities Date and Time bk Regional Preferences Contrast Version 1 0 O f Javon Select screen layout themes history and Baseline Compensation Adjust screen contrast amp brightness Serial number and software version Page 56 68 Miser 7 1 Date and Time The procedure is as follows Date and Time Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 7 2 Regional Sets Number Format The procedure is as follows Step 1 Regional Number Format
6. 5 3 Wavescan NanoPhotometer P Class User Manual An absorption spectrum can be obtained from your instrument enabling simple identification of peak height and position The procedure is as follows Parameter Screen wWavescan Parameters Step 1 Step 2 Start wavelength Pathlength Step 3 Step 4 End Wavelength Step 5 Hode gt OF E Cancel Step 7 Measurement Screen Step 8 2 5 Step 9 420 440 460 420 E00 a 450nm Sample Results Screen Wavescan I 0 30 j I 0 25 pofi 0 20 l pa 0 15 ka A 010A frl spear Ee 0 05 i l 400 Da az 6 aes 10 Abs 0 150 0 348 0 033 0 141 Sample 1 Parameter Screen Press 3 to select Functions Press 3 to select Wavescan Set Start Wavelength by using keypad numbers or left and right arrows Set End Wavelength by using keypad numbers or left and right arrows Select the Mode Absorbance or Transmission using the left and right arrows Select the Pathlength using the left and right arrows Options are 0 04 0 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications The Pathlength selection has no influence to the result It is just to document the used pathlength To enter the measurements screen with the selected parameters press OK D OR cancel the selections and return to the Functions folder by pressing Cancel Measurement Screen Insert the reference sample Press Blank key This will be used
7. Moser NanoPhotometer P Class User Manual P 300 P 330 P 360 Version 1 0 P 360 Maser telephone 800 227 9997 Fax 636 225 9998 Email custserv midsci com www midsci com Miser NanoPhotometer P Class User Manual Moser Implen GmbH Schatzbogen 52 D 81829 Germany Declaration of conformity for the NanoPhotometer P Class P300 P330 P360 This is to certify that the Implen NanoPhotometer P Class conforms to the requirements of the following Directives 2006 95 EC Low Voltage Equipment Safety Directive 98 79 EC In Vitro Diagnostic Medical Devices Directive 2004 108 EC EMC Directive 2002 95 EC Restrictions on the use of certain Hazardous Substances in Electrical and Electronic Equipment ROHS 1995 5 EC Radio and Telecommunications Terminal Equipment Directive instruments fitted with Bluetooth accessory only 2002 96 EC EC Directive on Waste Electrical and Electronic Equipment WEEE 2003 108 EC amp 2008 34 EC By ensuring this product is disposed of correctly you will help prevent potential negative consequences for the environment and human health which could otherwise be caused by inappropriate waste handling of this product Standards to which conformity is declared where relevant are as follows EN61010 1 2001 Safety requirements for electrical equipment for measurement control and laboratory use General requirements EN561010 2 101 2002 Particular requirements for IVD medical equi
8. the proper performance of a spectrophotometer has to be tested and proved on regular individually set intervals Implen provides certified NanoPhotometer P Class secondary standards as an optional accessory These NanoPhotometer P Class Didymiumglassfilters are suitable for the control and documentation of the wavelength accuracy and the photometric accuracy of your system IQ OQ documentation is also available only for P 330 and P 360 Please contact your local Implen office or an authorized Implen partner for further information Support agreements that help to fulfil the demands of regulatory guidelines concerning GLP GMP calibration certification using filters traceable to international standards during production and quality control certified engineers and calibrated test equipment approved to ISO 9001 standard automatic self diagnostic calibration test during start of the NanoPhotometer P Class result is documented in each data output file possibility to save a PVC file no data manipulation possible hardcopy printout 8 2 Lamp Replacement The xenon lamp should not need replacement until after several years of use In the unlikely event that it does need replacing this should be undertaken by a service engineer from your supplier 8 3 Mixer replacement The mixer should not need replacement until after several years of use In the unlikely event that it does need replacing this should be undertaken by a
9. OR press Back to return to the Standards screen Page 30 68 Moser Calibration Screen manual entry Lowry Calibration ru 0 017 A 0 056 4 a 0 094 A p 01328 ir 0 1694 tod m in a o 0 205 4 0 05 A nga 04 G6 QE LO LO LY K 2 Back EH oO Results screen r50 nm units asin Absorbance 0 035 4 Curve Fit 0 627 Regression Sample Pathlength Standard Pathlength NanoPhotometer P 300 Options select using key pad numbers Parameters Print Graph Edit Sample Pathlength Sample Number Save Method Printer Settings OY 8009 NanoPhotometer P 330 P360 Menu select using key pad numbers Sb Parameters gt Data Transfer Ch Edit Sample Pathlength Sample Number Save Method Output Options OO8 Version 1 0 Step 23 Step 24 Step 25 Step 26 Step 27 Step 28 NanoPhotometer P Class User Manual Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range is 0 001 to 9 999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Press OK 9 to accept the calibration and go to the Results screen see below
10. On off key Turns the instrument on off Arrow keys Use the four arrow keys to navigate around the display and select the required setting from the active highlighted option View options for that application mode Some of these are common to all View Options applications and described on page 8 Menu unique to an application are described in the relevant section of the NanoPhotometer P Class User Manual Use these to enter parameters and to write text descriptions where appropriate or required Use repeated key presses to cycle through lower case number and upper case Leave for 1 second before entering next character Use C button to backspace and 1 to enter a space Escape from a selection and return to the previous folder Cancel a selection Escape Cancel Back Stop making measurements Blank Reference Set reference to 0 000 A or 100 T on a reference solution at the current wavelength in the mode selected When in scan mode does a reference scan Sample Enter selection OK O Enter or confirm a selection Take a measurement Alphanumeric keys Version 1 0 Page 6 68 Miser NanoPhotometer P Class User Manual 2 4 Keypad and display for NanoPhotometer P 330 P 360 The back lit liquid crystal display is very easy to navigate around using the alphanumeric entry and navigation arrow keys on the hard wearing spill proof membrane keypad IMPLEN Print with Built in Printer ON Off Key Toggle Graph Data View Men
11. Toggle between Absorbance and T mode 4 Print graph greyed out if no data are available Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open Output Options settings possibility to change the Output settings within the method as described in 7 3 Output Options Printer 7 Exit Menu by pressing Escape Q or wait Page 39 68 Miser 5 2 Concentration NanoPhotometer P Class User Manual This makes simple concentration measurements on samples by measuring the amount of light that has passed through a sample relative to a reference this can be air Concentration is obtained by multiplying the measured absorbance at a specific wavelength by a factor The factor may be known in advance or may be calculated by the instrument by measuring a standard of known concentration The procedure is as follows Parameter Screen Concentration Parameters Wavelength a Pathlength T OF E Cancel E a Fa E Fa Cancel 3 saa aaa aaa aaa T AA Results Screen if using a Factor Concentration Version 1 0 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Parameter Screen Press 3 to select Functions Press 2 to select Concentration Set Wave
12. and Lid 250 0 04 mm can be ordered optionally The dilution factor lid factor is printed on the lid Please make sure that you use the appropriate lid for your sample 3 1 Technical instructions Step 1 Insert the NanoPhotometer P Class Submicroliter Cell into the cell holder with the cell windows facing the light beam We recommend facing the Implen logo to the front The light beam is directed from RIGHT to LEFT as indicated with small arrows Insert the NanoPhotometer P Class Submicroliter Cell always in the same direction Step 2 Use the integrated vortexer P 330 P 360 only to mix your sample well to achieve an accurate homogeneity of the sample Step 3 Pipette the appropriate sample volume onto the centre of the measuring window Warning Do not overfill the well lid Sampie volume Pathlengih 35 5 10 optional for P00 1 3 yl 03 2 am optional 0 3 2 yl T 100 250 optional 0 3 2 l i a Step 4 Make sure that for the measurements the lid fits exactly onto the positioning supports mounted to the body of the cell Take measurement Remember to consider the lid factor in your instrument software Please refer to the NanoPhotometer P Class User Manual for detailed information Step 4 Take the lid off and retrieve the sample with a pipette for further applications if desired Remove sample residues from the measurement window and the mirror in the lid Clean the measurement window and mirror in the l
13. each standard concentration point Can be Off 1 2 or 3 Step 11 Press Next to enter the Standards screen OR press Cancel Bio cancel selections and return to the Protein S ome basa Standards Screen Step 12 Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range 0 001 to 9 999 C button backspaces and clears the last digit entered 0 200 Step 13 Press Next gt to enter the Calibration screen If there are aes ee duplicate or non monotonic increasing entries the unit will beep and highlight the incorrect entry OR press Back to return to the Parameter screen Std 3 Std 6 et Mest i Back BCA Parameters Replicates Standards Screen BCA Standards Version 1 0 Page 23 68 Miser NanoPhotometer P Class User Manual Calibration Screen replicates off BCA Calibration Standards 0 700 0 400 0 600 Ls 0 200 Dha 04 D6 MB LO Le 14 BCA Calibration z x TETN B id DA l D3 0 4 0 6 0 8 1 0 Le 1 4 ok Calibration Screen replicates on BCA Calibration Replicates 1 DEZE 4 2 D6268 mg ia 1 400 0 626 A F 0 3 p Da Kal fa Dl by tE DH DE 0A LO Le Loy Version 1 0 Calibration Screen replicates off Step 14 This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all sub
14. 12 If the absorbance value of the sample is not in the linear range a Warning message will pop up and Instruction will be displayed in the top left corner of the result screen Please refer to 3 2 Software instructions important information on page 11 for further information Step 13 Repeat for all samples was ml Step 14 Press Menu Options to display available Options which are described on page 8 Step 15 Press Escape and confirm with Yes 9 to return to the Protein folder 0 359 A A280 0 012 4 0 003 4 TI To change parameters print or save methods press the Menu Options button The options menu will be opened For further explanation please see 2 3 Keypad and display on page 6 P 300 and 7 P330 P 360 Version 1 0 Page 20 68 Mioser 4 2 3 Protein UV Dye Method The procedure is as follows Parameter Screen NanoVolume Applications Protein Oye Parameters Lid Factor Protein Dilution Factor Units Ove Correction Cuvette Applications D Cancel Protein Dye Parameters Pathlength Protein Dilution Factor Units Oye Correction BS Cancel Protein Ove Oye Parameters Dye Abs Max Dye Ext Coefficient 500000 Dye Type Custom d Dve Correction B Cancel Version 1 0 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 NanoPhotometer P Class User Manual Parameter Screen Press 1 for NanoVolume OR 2 for Cuvette
15. 77 Abs max aye Abs 320 aye 108 Abs 260 Abs 320 With dye correction FOI 8 77 Abs max dye Abs 320 E dye 106 Abs 260 Abs 320 CF aye Abs max dye Abs 320 FormulaforRNA FOI 8 11 Abs max dye Abs 320 E aye 108 Abs 260 Abs 320 With dye correction FOI 8 11 Abs max dye Abs 320 E aye 108 Abs 260 Abs 320 CF aye ADS max dye Abs 320 FormulaforOligonucleotides FOI 9 83 Abs max dye Abs 320 E aye 108 Abs 260 Abs 320 With dye correction FOI 9 83 Abs max dye Abs 320 E dye 108 Abs 260 Abs 320 CF aye ADS max dye Abs 320 Abs max dye absorbance at absorption maximum of the dye AU E dye dye dependent extinction coefficient M1 cm1 The following dye types and parameters are pre programmed in the NanoPhotometer P Class Dye dependent extinction Dye dependent correction coefficient Eaye factor 260 nm CFopye Alexa Fluor647_ S SSS CSC BWOSC CN SCNSC C SCSNC UOUSOONSCSC CCN alexa Fluor 660_ 660 07 000 ow AexaFuor680_ 680 464 000 o0 og 880 280 000 oe Oysters 660 200000 00 In all formulas the molar dye dependent extinction coefficient is used Dye Type Absorption maximum Dye nm Version 1 0 Page 66 68 Miser NanoPhotometer P Class User Manual 10 3 Protein qu
16. 999 Molecular weight 0 001 to 9 999 999 Extinctioncoefficient I g cm Step 9 Ranges are Wavelength 200 nm to 340 nm Extinction coefficient 1 g cm 0 001 to 9 999 Select the Units of measurement using the left and right arrows Options mg ml ug ml ng ul and yug ul Step 10 Enter the protein dependent extinction coefficient Range is 10 000 to 9 999 999 Step 11 Press OK to store the chosen parameters and to enter the next screen OR Cancel Q to return to the Protein folder Step 12 Select the appropriate Dye Type 4 different AlexaFluors 2 Cy Dyes 2 DyLight Dyes FITC Pacific Blue r PE and Texas Red are programmed with their corresponding maximum absorbance wavelength dye dependent extinction co efficient and dye dependent correction factor at 280 nm For further details please refer to 10 4 Protein fluorescent dye incorporation Step 13 If using Custom Dye maximum absorbance wavelength of the custom dye dye dependent extinction coefficient and dye dependent correction factor at 280 nm have to be entered Ranges are Dye Abs Max 300 nm to 950 nm Dye Ext Coefficient 10 000 to 9 999 999 Dye Correction O to 0 999 Page 21 68 Miser Results Screen NanoPhotometer P Class User Manual m CEA Protein Dye A280 0 035 4 ADye 346 O 003 4 Protein Concentration 464 war mil Dye Concentration 0 00 pmol pl Degree of Labelling Results Screen Step 14 Apply inse
17. Multi wavelength Ar im F Ap Sbsorbance Ratio sanas Curse Absorbance or 96T transmission at a single user defined wavelength Colorimetric assay at a single wavelength based on a simple Factor entered or calculated from a single standard Spectral plot between two user defined wavelengths Range 200 950 nm with user configurable peak finding function Colorimetric assay at a single wavelength based on a user programmed curve Absorbance or T transmission at up to 5 user defined wavelengths Ratio of absorbance values at two user specified wavelengths Co Menu Options Within each function the user has the possibility to select various options that define the way results are treated If not using a stored method it is advisable to check that these options have been appropriately set for your experiment when coming to the instrument Note that setting the History parameter to On see Preferences later will cause the instrument to store its last settings If the History parameter is turned Off all parameters and selections will return to their default settings when leaving that application Unless it has been saved as a method Version 1 0 Page 37 68 Miser 5 1 Single Wavelength Abs and T NanoPhotometer P Class User Manual This makes simple absorbance A and transmission T measurements on samples measuring the amount of light that has passed through a sample relative
18. Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected So 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters OR Cancel Q Step8 Press Next 9 iv enter the next screen Step 9 Select the Calibration mode either Standards measure prepared standards Manual keypad data entry or New Standards previous values are blanked new standard can be measured Step 10 if Standards selected Select the number of Replicates using the left and right arrows This determines the number of standards to be measured and averaged at each standard concentration point Can be Off 1 2 or 3 Step 11 Press Next to enter the Standards screen OR press Cancel Bio cancel selections and return to the Protein folder Standards Screen Step 12 Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range is 0 001 to 9 999 C button backspaces and clears the last digit entered Step 13 Press Next to enter the Calibration screen If there are duplicate or non monotonic increasing entries the unit will beep and highlight the incorrect entry OR press Back o to return to the Parameter screen Page 26 68 Miser NanoPhotometer P Class User Manual Calibration Screen replicates off Bradford Calibration Standards 0 700 0 400 0 600 the 0 300
19. OR press Back to return to the Standards screen Results screen Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Insert the sample and press Sample O The concentration of the sample is taken and displayed Repeat for all samples Press Menu Options to display available Options which are described below press Escape and confirm with OK o to return to the Protein folder Query needs confirmation to avoid unintended escaping the application Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle graph on off Displays the calibration graph cursors give values for last measured sample 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape O or wait Menu select using key pad numbers Na Ne SNA NAN 1 2 4 T Return to parameters screen Transfer the results via selected Output Options Possibility to edit the sample pathlength Sample number add a prefix to the sample number and reset the
20. Options are 5 mm or 10 mm Enter the Dilution Factor using the keypad numbers Range 1 00 to 9 999 Use the C button to backspace and clear the last digit entered OR press Menu Options to enter the dilution factor screen Enter the volume of the sample using the keypad numbers Range 0 01 to 9 999 Enter the volume of the diluent using the keypad numbers Range 0 01 to 9 999 Press OK D to calculate the dilution factor and return to the Parameters screen OR press Cancel to cancel the selections and return to the Parameters screen Background correction at 320 nm is recommended to be switched On Select the Units of measurement using the left and right arrows Options ug ml ng ul ug ul Enter the Factor using the keypad numbers Default value is 50 for dsDNA 37 for ssDNA and 40 for RNA range is 0 01 to 9 999 Press OK to enter the Results screen OR Cancel to return to the Nucleic Acids folder Results Screen Apply insert the reference sample Press the Blank Key This will be used for all subsequent samples until changed Apply insert sample and press Sample This measures at the selected wavelengths and displays the results The sample concentration the ratio of A260 A280 and A260 A230 are calculated corrected by the background wavelength value if selected If the absorbance value of the sample is not in the linear range a Warning message will pop up and Instruction will be displayed in the top left co
21. Save Method Output Options a O o o O Version 1 0 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad Step 23 The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range is 0 001 to 9 999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Step 24 Press OK to accept the calibration and go to the Results screen see below OR press Back G to return to the Standards screen Results screen Step 25 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 26 Insert the sample and press Sample O The concentration of the sample is taken and displayed Step 27 Repeat for all samples Step 28 Press Menu Options to display available Options which are described below Step 29 Press Escape O and confirm with Yes to return to the Protein folder Query needs confirmation to avoid unintended escaping the application Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle graph on off Displays the calibration graph cursors give values for last measured sample 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing n
22. Standard volume applications can be performed with 10 mm pathlength quartz glass or plastic cuvettes Note Within the Utilities folder the user has the possibility to select various options that define data output please see also 7 3 Output Options Printer The NanoVolume Applications folder and the Cuvette Applications folder contain different sub folders Folder Application Recommended Measurement Cell Nucleic Acids DNA Concentration purity check and dye incorporation for Submicroliter Cell Cuvette DNA samples RNA Concentration purity check and dye incorporation for Submicroliter Cell Cuvette RNA samples Oligo Concentration purity check and dye incorporation for Submicroliter Cell Cuvette Oligo samples Protein Protein UV Christian Warburg Protein determination at 280 nm Submicroliter Cell Cuvette Protein Dye Protein determination at 280 nm and dye incorporation Submicroliter Cell Cuvette BCA Protein determination at 562 nm Cuvette Bradford Protein determination at 595 nm Cuvette Lowry Protein determination at 750 nm Cuvette Biuret Protein determination at 546 nm Cuvette Cell Count OD600 Cell density at 600 nm Cuvette Nucleic Acids Protein and OD 600 Cell Density Contents of these sub folders are detailed below 4 1 Characterization of DNA RNA and Oligonucleotides 4 1 1 General Information Nucleic Acid Quantification NAQ Nucleic acids can be quantified at 260 nm because it is well established that a
23. Standby mode after defined periods Options are 1 hour 2 hours at night or off Select Baseline Compensation to improve value stability and to overcome background effects Press OK to store the settings and return to the Utilities folder OR press Cancel to return to the Utilities folder without storing the settings Ambient temperature can affect the display This function can optimize the display for local conditions The procedure is as follows Step 1 Step 2 E Ka Version 1 0 Adjust the Brightness using the left and right arrows Adjust the Contrast using the left and right arrows Press OK d to store the settings and return to the Utilities folder Page 58 68 Miser NanoPhotometer P Class User Manual 7 6 About About NMancPhotometer Displays the instrument serial number and software version Senai Rumke 54321 Press OK Con to close the window and return to the Utilities folder Yersion T122 v2 2 0 or wait Build 11 Wvww_implen de cc OK Version 1 0 Page 59 68 Miser NanoPhotometer P Class User Manual 8 MAINTENANCE 8 1 Maintenance free Technology The NanoPhotometer P Class technology is maintenance free Regular maintenance or calibration is not necessary For facilities that are working according to national as well as international guidelines and standards such as Good Laboratory Practice GLP Good Manufacturing Practice GMP or ISO9000 9004
24. access a list of pre defined units press the Menu Options key and then use the left right arrows ug ml ug ul pmol ul mg ml mmol l umol 1 g l mg l ug l U I Yo ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed Decimal Points DP to be selected from O to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters OR Cancel Q Press Next Oio enter the next screen Select the Calibration mode either Standards measure prepared standards Manual keypad data entry or New Standards previous values are blanked new standard can be measured if Standards selected Select the number of Replicates using the left and right arrows This determines the number of standards to be measured and averaged at each standard concentration point Can be Off 1 2 or 3 Press Next Yi enter the Standards screen OR press Cancel to cancel selections and return to the Protein folder Standards Screen Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range is 0 001 to 9 999 C button backspaces and clears the last digit entered Press Next D io enter the Calibration screen If there are duplicate or non monotonic
25. and press sample No changes Do you want to change the lid factor automatic change of lid factor lid 100 to lid 50 in Concentration too high Dilute sample or change to lid 250 the software for calculation Lid 250 Concentration too low Please change to lid 100 and press sample No changes Do you want to change the lid factor automatic change of lid factor lid 250 to lid 100 in Concentration too high Dilute sample Lid volume Warning message Instruction eee a ol Some of P360 is Li Version 1 4 abs GTIN Sample concentration is too low Ele is too high D orange to lid E fon a Sample concentration is too low or change to lid 5 if e klabs too low available Ele S too high FErvstcotow cnange to a ka Abs is too high Abs is too high Physical dilution of the sample is necessary or change to lid 250 if E Ess t0 10w Abs is too high Physical dilution of the sample is necessary the lids are only optional available Lid delivery content for P 300 is Lid 50 for P 330 Lid 10 and Lid 50 and for d 10 Lid 50 und Lid 250 O Page 11 68 Miser NanoPhotometer P Class User Manual 4 NANOVOLUME APPLICATIONS AND CUVETTE APPLICATIONS The NanoPhotometer P Class offers a complete solution for NanoVolume and standard volume applications With the NanoPhotometer P Class Submicroliter Cell the required sample volume ranges from 0 3 ul to a maximal sample volume of 5 ul
26. and store the result Step 22 Repeat for all replicates and standards A graph will display the results and the fitted curve as the measurements are input Step 23 Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 24 Press Next to accept the calibration and go to the Results screen see below OR press Back o to return to the Standards screen Page 48 68 Miser NanoPhotometer P Class User Manual Calibration Manual entry Standard Curve Calibration 430 nm units asin Parameters Data Transfer Edit Sample Pathlength Sample Number Save Method Output Options Version 1 0 Calibration Manual entry Step 25 Shows previously entered calibration values and allows values to be entered via the keypad Step 26 The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9 999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Step 27 Press OK O to accept the calibration and go to the Results screen see below OR press Back to return to the Standards screen Results screen Step 28 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 29 Insert the sample and press Sample gt The
27. concentration of the sample is taken and displayed Step 30 Repeat for all samples Step 31 Press Menu Options to display available options which are described below Step 32 Press Escape Q and confirm with Yes gt to return to the Functions folder Query needs confirmation to avoid unintended escaping the application Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options Printer settings 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open Output Options Printer settings possibility to change the Output Printer settings within the method as described in 7 3 Output Options Printer Exit Menu Options by pressing Escape or wait Page 49 68 Miser NanoPhotometer P Class User Manual 5 6 Multiple Wavelength This makes up to 5 absorbance measurements on the same sample The procedure is as follows Parameter Screen Parameter Screen Multi wavelength Parameters Step 1 Press 3 to select Functions Step2 Press 6 to select Multi Wavelength Step3 Select the number of Wavelengths Step4 Select the Pathlength using the left and right arrows Options are 0 04 0 1 0 2 1 and 2 mm for NanoVolume applications
28. for all subsequent samples until changed Insert sample and press Sample gt Step 10 Repeat for all samples Results Screen A graph of the wavescan is displayed along with a table of Absorbance T at each peak Up to eight peaks can be shown Use the left and right arrows to move the cursor along the graph When it reaches a peak the peak height and width of the peak is displayed at the top of the screen To zoom in on the wavelength scale use the up arrow This auto scales on the Absorbance T scale dependent on the Graph Scale option and this is retained for subsequent measurements To zoom out again use the down arrow Step 11 Press Menu Options to display available options which are described next Step 12 Press Escape Q and confirm with Yes gt to return to the Version 1 0 Functions folder Query needs confirmation to avoid unintended escaping the application Page 42 68 Miser NanoPhotometer P Class User Manual Menu select using key pad numbers Menu select using key pad numbers 1 Return to parameters screen pig kaaa 2 Mhal the results via selected Output Options Printer settings 3 d 8T l 3 Toggle between Absorbance and T mode Peak Detection 4 Displays Peak Detection Parameter Screen See description below Graph Scale 5 Manually adds a peak position to the peak table in the results Sample Number screen at the position set by the cursor If the cursor is retu
29. increasing entries the unit will beep and highlight the incorrect entry OR press Back O to return to the Parameter screen Page 32 68 Miser NanoPhotometer P Class User Manual Calibration Screen replicates off Biuret Calibration Standards 0 700 0 400 0 600 0 300 Biuret Calibration Standards 0 200 0 044 4 Lo 0 5 Dha 04 GE OB LO Le L4 2 Back 0 400 0 1484 Ha 0 600 0 245 A iy 0 800 0 3494 0 4 ra Fa 1 000 0 446 4 a A 1 400 0 542 4 A Dl mi no O14 E MA LO Lo Ly ABA Calibration Screen replicates on Biuret Calibration Replicates 2 0 345 4 Version 1 0 1 0 3492 Big 0 800 0 345 A oA ja ne Ki Fa Ol si Da 0 4 D6 0 8 LO Le 14 2 Back Step 14 Step 15 Step 16 Step 17 Step 18 Step 19 Step 20 Step 21 Step 22 Calibration Screen replicates off This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Insert the standard use C to clear previously stored results before measuring Press Sample to measure the standard and store the result Repeat for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading W
30. molar extinction coefficient M cm or 1 extinction coefficient I g cm In the software are the following protein A280 factors pre programmed BSA bovine serum albumin serum albumin mouse and human lysozyme IgG and OD 1 for more information about the factors see 11 3 Protein quantification There is also the possibility to enter custom factors For correct calculation the following settings are needed either the extinction coefficient I g cm or the molar extinction coefficient M14cm1 and the molecular weight g mol of the protein Rapid measurements such as this at 280 nm are particularly useful after isolation of proteins and peptides from mixtures using spin and HiTrap columns by centrifuge and gravity respectively Protein determination at 280 nm and degree of labelling NanoVolume Applications and Cuvette Applications To determine the degree of labelling the absorbance reading at the wavelength reported for maximum absorbance of the fluorescence dye is used The corresponding extinction coefficient of the dye is used in the Lambert Beer Law to determine the dye concentration c A e d Absorbance values and extinction coefficients are used to calculate the dye per protein ratio For further details please refer to 10 4 Protein fluorescent dye incorporation Colorimetric Bradford Biuret BCA and Lowry protein determination Cuvette Applications The Bradford method depends on quantifying the binding of a dye Coomass
31. printed out A correlation coefficient of between 0 95 and 1 00 indicates a good straight line Version 1 0 Page 18 68 Miser NanoPhotometer P Class User Manual 4 2 2 Protein UV Method The procedure is as follows Parameter Screen Parameter Screen NanoVolume Applications Step 1 Press 1 for NanoVolume OR 2 for Cuvette folder Step 2 Press 2 to select Protein folder Lid Factor A Step 3 Press 1 to select Protein UV mode Step 4 Using NanoVolume Applications select the Lid Factor as described in the Average Detection Range Sheet or under Dilution Factor Toe 3 2 A minimum of 1 5 ul sample volume for lid 10 is recommended Using Cuvette Applications select Pathlength using the left and right arrows Options are 5 mm or 10 mm Step 17 Enter the Dilution Factor using the keypad numbers Range Protein UY Parameters 1 00 to 9 999 Use the C button to backspace and clear the n last digit entered OR press Menu Options to enter the dilution factor screen Enter the volume of the sample using the keypad numbers Range 0 01 to 9 999 Enter the volume of the diluent using the keypad numbers Range Cuvette Applications 0 01 to 9 999 Press OK O to calculate the dilution factor and return to the Parameters screen OR press Cancel Q to cancel the selections and return to the Parameters screen Step 5 Select whether the Background correction at 320 nm is used or not with the left and right arrows It is recommended to switch
32. service engineer from your supplier Caution Do not open the base plate of the vortexer Do not break the seal 8 4 Exchange of the gaiter If the gaiter is damaged please exchange it immediately The gaiter can be ordered separately item number is S 330360 G Caution Switch the instrument off before removing the gaiter Do not look into the gap between the mixer and the housing UV exposure 1 Remove the defect gaiter Version 1 0 Page 60 68 Miser NanoPhotometer P Class User Manual 2 Place the new gaiter in the gap and in a first step place inner flute and in the second step the outer 8 5 Cleaning and general care of the instrument 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 NanoPhotometer P Class design edition glossy anthracite All painted surfaces of the NanoPhotometer P Class can be cleaned with a soft damp cloth and approved cleaning or disinfectant solutions Caution Product damage by wrong cleaning or disinfection Desinfection or cleaning only by wiping no spraying Use no solvents or aggressive chemicals Approved disinfectant solutions Apesin disinfection spray Tana Chemie GmbH Incidin Liquid and Incidin Foam Ecolab Lysoformin Spezial Lysoform Dr Hans Rosemann GmbH Observe all necessary precautions if dealing with hazardous
33. to a reference this can be air The procedure is as follows Parameter Screen Step 1 Step 2 Step 3 Single wavelength Parameters Wavelength Step 4 Mode Pathlength gt Step 5 OF E Cancel Step 6 Results Screen Single wavelength Step 7 si A 450 nm m imn o Resat Step 9 Units x o 460 410 Single wavelength Abs Version 1 0 Parameter Screen Press 3 to select Functions Press 1 to select Single Wavelength Set Wavelength by using keypad numbers or left and right arrows Select the Mode Absorbance or Transmission using the left and right arrows Select the Pathlength using the left and right arrows Options are 0 04 0 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications The Pathlength selection has no influence to the result It is just to document the used pathlength To enter the results screen with the selected parameters press OK OR cancel the selections and return to the Functions folder by pressing Cancel Q Results Screen Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Insert sample and press Sample NY Repeat for all samples Step 10The result at the selected wavelength is displayed on the screen Step 11 Use the left and right arrows to move the cursor and display the value at the cursor position 15nm from set wavelength Step 12 P
34. 0 0 410 0 Wavelength nm Note absorbance maximum near 260 nm and absorbance minimum near 230 nm flat peak near 260 nm and steep slope at 280 nm very little absorbance at 320 nm Operation of the instrument for Nucleic Acid measurements is described in the following sections DNA and RNA are very similar whilst in Oligo it is possible to calculate the factor from the composite bases by entering the proportions of the 4 bases Version 1 0 Page 13 68 Miser 4 1 2 Analysis of dsDNA ssDNA and RNA The procedure is as follows Parameter Screen NanoVolume Applications AsOMA Parameters Lid Factor Units Dilution Factor Factor Background Cuvette Applications AsONA Parameters Pathlength Units Dilution Factor Factor Background B Cancel o Cancel Results Screen A280 0 001 A AZ60 AZ80 AZEBOSAZIO 3 241 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step9 Step 10 Step 11 Step 12 Step 13 Step 14 Step 15 NanoPhotometer P Class User Manual Parameter Screen Press 1 for NanoVolume OR 2 for Cuvette folder Press 1 to select Nucleic Acids folder Press 1 to select dsDNA mode OR 2 to select ssDNA mode OR 3 to select RNA mode Using the NanoVolume Applications select the Lid Factor as described in the Average Detection Range Sheet and under 3 2 Using Cuvette Applications select Pathlength using the left and right arrows
35. 0 for concentration use a reagent blank when required to enter the zero standard The procedure is as follows Parameter Screen Step 1 Step 2 Step 3 Standard Curve Parameters Wavelength Pathlength 430 nm Step 4 Standards Step 5 Units 2 Cancel Step 6 Standard Curve Parameters Step 7 Standard Curve Parameters Curve Fit 4 Regression Step 8 Calibration Replicates E Hent Step 9 Cancel Version 1 0 Parameter Screen Press 3 to select Functions Press 5 to select Standard Curve Select the Wavelength using the keypad numbers or left and right arrows Enter the number of Standard concentration points to be used in the curve 1 9 Select the Pathlength using the left and right arrows Options are 0 04 0 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications The Pathlength selection has no influence to the result It is just to document the used pathlength Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Menu Options key and then use the left right arrows ug ml ug Ul pmol ul mg ml mmol l umol I g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed Decimal Points DP to be selected from O to 2 Note that the result will always be fixed to 5 significant figures rega
36. 2 to select BCA mode 562 nm Step4 The default Wavelength setting is 562 nm Step 5 Enter the number of Standard concentration points 1 9 RRE to be used in the curve using the keypad numbers or left Ps ariin ee Step 6 Select Pathlength using the left and right arrows Options are 5 or 10 mm Units Cont Step 7 Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Menu Options key and then use the left right arrows ug ml ug ul pmol ul mg ml mmol l umol 1 g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed Decimal Points DP to be selected from O to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected So 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters OR Cancel BCA Parameters AAAH ea Sao as nnan Step 8 Press Next D iv enter the next screen Step 9 Select the Calibration mode either Standards measure prepared standards Manual keypad data entry or New Standards previous values are blanked new standard can urve FI be measured Step 10 if Standards selected Select the number of Replicates using the left and right arrows This determines the number of standards to be measured and averaged at
37. 280 nm to be delivered from dye supplier E dye dye dependent molar extinction coefficient M1 cm3 The following dye types and parameters are pre programmed in the NanoPhotometer P Class Absorption maximum Dye dependent correction Dyes nm coefficient Eaye factor 280 nm CFoye os lss lc tago 008 Cy Dumas s gogo In all formulas the molar dye dependent and the molar protein dependent extinction coefficient is used Version 1 0 Page 68 68
38. 4 999 5 Step 2 E Cancel 7 3 Output Options Printer Sets up Printer settings P300 The procedure is as follows Step 1 Auto Print Oo a Printer Step 2 O E Cancel Step 3 Version 1 0 NanoPhotometer P Class User Manual Enter the Day using the keypad numbers or left and right arrows Enter the Month as above Enter the Year Enter the Hour Enter the Minute Seconds are zeroed when OK is pressed Press OK O to store the settings and return to the Utilities folder OR press Cancel Q to return to the Ultilities folder without storing the time Set the Number Format decimal point style Options are kb 77 kb 17 or Press OK o to store the settings and return to the Utilities folder OR press Cancel to return to the Utilities folder without storing the settings Select whether Auto print is on or off using the left and right arrows When auto print is on the results are automatically printed after a measurement is taken When it is off printing has to be initiated manually This can also be set using the Options key in each application or method The default is OFF Select how the data are sent Options are Computer USB and depending on the attached printer module Built in SD Memory Card E or Bluetooth This can also be set using the Options key in each application or method Press OK Od to store the settings and return to the Utilities folder OR Pr
39. 8 Miser NanoPhotometer P Class User Manual 11 SPECIFICATION AND WARRANTY Technical Specifications Spectrophotometer Wavelength range 190 1 100 nm Wavelength scan range 200 950 nm System start up time Less than 5 seconds no warm up necessary Measure time for full scan 3 5 seconds range lt 0 5 at 220 nm using Nal and 340 nm using NaNO2 0 003 A O to 0 5 A 0 007 A 0 5 1 0 A 260 nm 0 002 Arms at 0 A 260 nm 0 005 A pk to pk at O A 260 nm 15 mm centre height z height outside dimension 12 5 mm x 12 5 mm NanoVolume application Other technical data Nucleic acid microarray labelling efficiency protein and cell density 140 mm x275 mmx 380 mm CA Operating voltage voltage Operating voltage 90 250 V 50 60 Hz Max 30 VA esse 250V 90 250V 50 60Hz Max30VA dn Hz Max 30 VA Input Output ports SD Memory Card USB or Bluetooth for connection to a PC for direct data download printout and data storage Performance verification Auto diagnostics when switched on Specifications are measured after the instrument 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 Warranty IMPLEN guarantees that the product supplied has been thoroughly tested to ensure that it meets its published specification The warranty included in the condit
40. Abs 320 CF dye Abs max dye Abs 320 A280 factor lid factor dilution factor C prot protein concentration mg ml Abs 280 absorbance AU of proteins CF dye dye dependent correction factor at 280 nm to be delivered from dye supplier ADS max dye absorbance at absorption maximum of the dye AU A280 factor molecular weight prot molar extinction coefficient M14cm 1 prot oder 1 extinction coefficient 1 g cm lid factor virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid Calculation of fluorescence dye concentration pmol ul C dye ADS max dye Abs 320 lid factor dilution factor aye 10 4 C dye dye concentration pmol ul ADS max dye absorbance at absorption maximum of the dye AU lid factor virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid E dye dye dependent molar extinction coefficient M1 cmt Version 1 0 Page 67 68 Miser NanoPhotometer P Class User Manual Calculation of degree of labelling D P degree of labelling Abs max dye Abs 320 prot Abs 280 Abs 320 dye With dye correction degree of labelling Abs max dye Abs 320 prot Abs 280 Abs 320 Abs max dye Abs 320 CF aye dye ADS max dye absorbance at absorption maximum of the dye AU E prot protein dependent molar extinction coefficient M1 cm1 CF aye dye dependent correction factor at
41. O OR cancel the selections and return to the Functions folder by pressing Cancel O Results Screen if using a Factor Step 10 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 11 Insert sample and press Sample gt Page 40 68 Miser NanoPhotometer P Class User Manual Results Screen if using standard mode Concentration Run Standard Concentration Concentration wavelength 260 nm Absorbance 0 042 4 2571 NanoPhotometer P 300 Options select using key pad numbers Farameters Print Graph Run Standard Sample Number Save Method Printer Settings 600 S606 NanoPhotometer P 330 P 360 Menu select using key pad numbers Parameters Data Transfer Run Standard Sample Number Save Method Output Options 600 08 Version 1 0 Results Screen if using standard mode Step 12 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 13 Press Sample to display the Run Standard screen Step 14 Run the standard by pressing Run OR Press Cancel Q to return to the measure screen Step 15 Insert the sample and press Sample O The concentration of the sample is displayed Results shown as indicate the concentration is out of range Step 16 Repeat for all samples Step 17 Press Menu Options to display av
42. ODUONS PINE AA AA 41 Wee AA AA AA AA AA AA 41 Lo CONG NAA AA 41 AE AA AA AA AA AA AE AA AAP ANA BA 41 MANLENANG Bam AGA AA 41 8 1 Maintenance free Technology 1 manaa an naaaa aan KEBAA NN NANANA NNAANAN ANAN EAA 41 8 2 Lamp Replace men AANI AA AA 41 O MIXEEKECLIACEMEN NAAN AA AA AA 41 B4 Exchange olihe gahl cecainctccscoemensceatesencasixauonncnneeetecsinenteecaesececeunanesasusnaneusstnxseamcantsnesensauanetaecnssanseceen 41 8 5 Cleaning and general care of the instrument ssssnnnnunnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnna 41 GOO EnO NC SS aC S AA AA AA AA AA 41 Or WROUNIS SION AA AA AA AA 41 ACC O ODI a AA AO AA NA AA AA E 41 APPEAR AKA E AA AA AA AA 41 10 1 INUCICIC acid QuanUN GANON aa AA 41 10 2 Nucleic acid fluorescent dye incorporation 7 aasasasamaaaa na uEa nn NN NAE ANN KAKANAN NANANA NNN KAKANAN 41 10 3 Protem guantitaUON aa AA 41 10 4 Protein fluorescent dye incorporation asaasamaaa namun NN ANAN NANANA NANANA NA KANAN AN NANA NANA NNN NANANA NADAAN NAA 41 Version 1 0 Page 3 68 Miser NanoPhotometer P Class User Manual 1 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 AN Caution refer to acc
43. Remove cuvette or submicroliter cell from the cell holder turn off the instrument and start again Did you start the NanoPhotometer P Class directly after delivery If yes turn it of and wait 30 min before switching the unit on If both suggestions doesn t help disconnect the power completely for at least 10 seconds and then reconnect and restart the system again Remove cuvette or submicroliter cell from the cell holder turn off the instrument and start again Remove cuvette or submicroliter cell from the cell holder turn off the instrument and start again Remove the cell holder and place back in the right position If both suggestions doesn t help disconnect the power completely for at least 10 seconds and then reconnect and restart the system again Disconnect the power for quite some time to make sure that the problem is not occurring due to low energy sent to the Xenon flash lamp Disconnect the power completely for at least 10 seconds and then reconnect and restart the system again Check all connections if the cables etc are sticking right Check correct power supply 18 V is attached If possible try another power supply Please contact the Implen Support Team support implen de Phone 49 89 7263718 20 if none of the mentioned solution helps to solve the problem or if another error message should appear on the NanoPhotometer P Class display Version 1 0 Page 62 68 Miser NanoPhotometer P Class
44. User Manual 8 Trouble shooting Symptom tr ptom Symptom o iY Solution Instrument failing start up calibration AI ELE SO that the correct power supply 18 V 1 2 Ais being used and ensure that the connector is pushed in fully Instrument switching off after calibration User may be keeping their finger on the ON OFF button too long so that the instrument receives both signals and switches off after the calibration Instrument intermittently switches off during Faulty or loose power input connection Check voltage output of measurement power supply Thermal paper printer displays random or Paper roll was maybe changed when the instrument is still in one of endless numbers after paper exchange the programs User needs to move to the main menu before exchanging the paper roll Switch the device Off and disconnect the power supply reconnect it after about 5 minutes Firmware update version 2 3 recommended Thermal paper printer prints a plenty of paper Thermal Printer paper was probably put the wrong way check correct normal sound no error message position of the printer paper Thermal printer automatic roller stops Please contact the Implen support team Please contact the Implen Support Team Support implen de Phone 49 89 7263718 20 if none of the mentioned solution helps to solve the problem or if another symptom should occur 9 ACCESSORIES photometric accuracy for the NanoPhotometer Version 1 0 Page 63 6
45. ailable options which are described below Step 18 Press Escape Q and confirm with Yes to return to the Functions folder Query needs confirmation to avoid unintended escaping the application Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggles on off displaying a graph of wavescan 20 nm from selected wavelength 4 Return to Run Standard screen 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape O or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options 4 Return to Run Standard screen 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open Output Options settings possibility to change the Output settings within the method as described in 7 3 Output Options Printer Exit Menu by pressing Escape 1 or wait Page 41 68 Miser
46. and 5 or 10 mm for cuvette applications The Pathlength selection has no influence to the result It is just to document the used pathlength Step 5 Press OK O to enter the next screen Step 6 Enter the first Wavelength using either the number keys or Goo Cancel the left and right arrows Step 7 Enter the second Wavelength as above and repeat for the number of wavelengths selected up to 5 Step8 Press OK to enter the results screen OR press Cancel a4 to return to the Functions folder Pathlength Multi wavelength Parameters cy Ok Cancel Results Screen Multi Wavelenath Step 9 Insert the reference sample Press Blank key This will be f UU Sample O used for all subsequent samples until changed po Step 10 Insert sample and press Sample A Abs a 300 nm Step 11 Repeat for all samples A scan plot covering the range of wavelengths selected with cursors at the relevant wavelengths and a table of values is displayed 500 nm Step 12 Press Menu Options to display available options which are 400 nm described below 600 nm ne eas Step 13 Press Escape and confirm with Yes to return to the Functions folder Query needs confirmation to avoid unintended escaping the application Di yt P E sonm N GUO rir 0 013 my I il l i bi 800 nm 0 013 LOAN AEE ir a AN a ID 0 0 an 400 saa EE Version 1 0 Page 50 68 Miser NanoPhotometer P 300 Option
47. ange 1 00 to 9 999 Use the C button to backspace and clear the last digit entered OR press Menu Options to enter the dilution factor screen Enter the volume of the sample using the keypad numbers Range 0 01 to 9 999 Enter the volume of the diluent using the keypad numbers Range 0 01 to 9 999 Press OK to calculate the dilution factor and return to the Parameters screen OR press Cancel Q to cancel the selections and return to the Parameters screen Background correction at 320 nm is recommended to be switched on Select the Units of measurement using the left and right arrows Options ug ml ng ul ug ul and pmol ul Enter the Factor using the keypad numbers Default value is 33 range is 0 01 to 9 999 If pmol ul is selected there are two options to set the factor 1 A selection table denoting the ratios of the 4 bases according to the oligo sequence Enter the proportions of bases present using the keypad numbers and up and down arrows to move between boxes Default is 10 for each range is O to 9 999 2 Enter the known extinction factor of the oligo used factor range 0 01 to 9 999 for ratio 1 extinction coefficient x106 Press OK O enter the Results screen OR Cancel to return to the Nucleic Acids folder Results Screen Apply insert the reference sample Press Blank Key This will be used for all subsequent samples until changed Apply insert sample and press Sample This measures at the selected wavelengths and dis
48. antification For determination of protein concentration in solution the absorbance at wavelength 280 nm is used The function describing the concentration to absorbance relation is a modification of the Lambert Beer equation C prot Abs 280 A280 factor lid factor dilution factor With background correction C prot Abs 280 Abs 320 A280 factor lid factor dilution factor C prot protein concentration mg ml Abs 280 absorbance AU of proteins A280 factor Default setting is BSA molecular weight prot molar extinction coefficient M 4 cm prot oder 1 extinction coefficient I g cm Abs 260 absorbance AU of nucleic acids lid factor virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid A280 factors pre programmed Serum albumin Serum albumin BSA IgG L mouse human aaah alae R280 factor O71 038 Molecular Weight g mol 69 323 4 68 692 5 69 365 7 150 000 14 300 Molar extinction coefficient M1 cm4 47 790 43 780 39 310 210 000 37 500 10 4 Protein fluorescent dye incorporation To determine the protein concentration and the dye concentration after labelling a modification of the Lambert Beer equation is used Background correction is always calculated possibility to switch the dye correction on and off Calculation of labelled protein concentration C prot Abs 280 Abs 320 A280 factor lid factor dilution factor With dye correction C prot Abs 280
49. are OD or cells ml If cells ml is selected two further parameters are displayed if cells ml selected Enter the Factor using the keypad numbers Range 0 00 to 9 999 C button backspaces and clears the last digit entered if cells ml selected Select the Multiplier using the left and right arrows Options are 1 000 or 1 000 000 Factor and Multiplier define the conversion of the measured OD to the number of cells per millilitre e g 1 OD 600 5 x 108 cells ml Press OK to enter the Results screen OR press Cancel to cancel selections and return to the Cuvette Applications folder Results Screen Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Insert the sample and press Sample The wavelength absorbance and OD600 value is displayed Repeat for all samples Press Menu Options to display available Options which are described below Press Escape and confirm with Yes D iv return to the Cuvette Applications folder Query needs confirmation to avoid unintended escaping the application To change parameters print or save methods press the Menu Options button The options menu will be opened For further explanation please see 2 3 Keypad and display on page 6 P 300 and 7 P 330 P 360 Version 1 0 Page 36 68 Miser NanoPhotometer P Class User Manual 5 FUNCTIONS Survey of the available Functions Functions oA Single wavelength olat
50. beam is directed from RIGHT to LEFT through the cell chamber therefore please ensure the measurement cell is inserted in the correct alignment Insert the measurement cell always in the same direction The cell holder supplied with the instrument accepts the NanoPhotometer P Class Submicroliter Cell and standard 10 mm pathlength quartz glass or plastic cells The optical height of the NanoPhotometer P Class is 15 mm The minimum volume that can be used is 0 3 ul with the NanoPhotometer P Class Submicroliter Cell 12 mm test tubes may be used e g for cell cultures however they are not recommended as higher quality data is produced by using disposable cuvettes for the analysis If used align the indicator line on 12 mm test tubes in the same direction to ensure reproducible positioning of the tube Note that test tubes do not last forever and that the surface becomes scratched and blemished through repetitive use if this is the case they should be replaced Version 1 0 Page 5 68 Miser NanoPhotometer P Class User Manual 2 3 Keypad and display for NanoPhotometer P 300 The back lit liquid crystal display is very easy to navigate around using the alphanumeric entry and navigation arrow keys on the hard wearing spill proof membrane keypad IMPLER LCD Display ON OFF key NANOPHOTOMETER Alphanumeric keys Cellholder Escape Cancel Back Arrow keys Blank Reference Sample Enter selection OK View options
51. boxes Range 0 001 to 9 999 Step 12 Press Next to enter the Calibration screen If any duplicate or non monotonic increasing entries are present the unit will beep and highlight the incorrect entry OR press Back O to return to the Parameter screen Calibration Screen replicates off Step 13 This shows the calibration values and allows standards to be measured Step 14 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 15 Insert the standard use C to clear previously stored results before measuring and press Sample to measure the standard and store the result Step 16 Repeat for all standards A graph will display the results and the fitted curve as the measurements are input Step 17 Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 18 Press OK 9 to accept the calibration and go to the Results screen see below OR press Back O to return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Step 19 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 20 Press Replicates to display the replicate entry boxes Use C to clear previously stored results before measuring Step 21 Insert the standard and press Sample 6 to measure the standard
52. dards screen Page 24 68 Miser NanoPhotometer P Class User Manual Calibration Screen manual entry BCA Calibration 0 051 4 0 171 4 0 2884 0 404 A O516A 0 627 A ne ny x Dl ra Da 0 4 D6 0 8 LO Le 14 K E Back e Results screen 562 nm MUST Absorbance O 255 A Curve Fit Regression Standard Pathlength Sample Pathlength NanoPhotometer P 300 Options select using key pad numbers Parameters Print Graph Edit Sample Pathlength Sample Number Save Method Printer Settings O09 8300 NanoPhotometer P 330 P360 Menu select using key pad numbers lt gt Parameters Eb D ata Transfer Ch Edit Sample Pathlength Sample Number Save Method Output Options OO8 Version 1 0 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad Step 23 The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9 999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Step 24 Press OK to accept the calibration and go to the Results screen see below OR press Back G to return to the Standards screen Results screen Step 25 Insert the reference sample and press Blank key This will be used for all subse
53. e number of Replicates using the left and right arrows This determines the number of standards to be measured and averaged at each standard concentration point Can be Off 1 2 or 3 Press Next 9 enter the Standards screen OR press Cancel to cancel selections and return to the Protein folder Standards Screen Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range 0 001 to 9 999 C button backspaces and clears the last digit entered Press Next D i enter the Calibration screen If there are duplicate or non monotonic increasing entries the unit will beep and highlight the incorrect entry OR press Back O to return to the Parameter screen Page 29 68 Miser NanoPhotometer P Class User Manual Calibration Screen replicates off Lowry Calibration Lowry Calibration 1 400 0 205 4 2 m k Dha 04 C6 LB LO Le Ll4 0 200 0 0174 E 0 400 0 056 4 L O15 ff 0 600 0 095 4 ef 0 300 0 1324 Fa 10 pug 1 000 0 163 4 P Pi 4 105 pi nG 04 ME MA LO Lo Li Back Calibration Screen replicates on Lowry Calibration Mean Version 1 0 w sm m5 y ao A a r na 04 AE DA LO tLe LY E Back Step 14 Step 15 Step 16 Step 17 Step 18 Step 19 Step 20 Step 21 Step 22 Calibration Screen replicates off This shows the calibration values and allows standard
54. erence per se For this reason the change in absorbance per minute AA min concentration AA min x factor and correlation coefficient calculated from a best fit of the data points are displayed They may not be relevant for simple kinetics experiments The procedure to define a new method is as follows Parameter Screen Parameter Screen Kinetics Parameters 1 Step 1 Press 3 to select Functions Step 2 Press 4 to select Kinetics Step 3 Wavelength Enter all numerical values using the keypad numbers or the left and right arrows Step 4 Delay time Enter the delay time in seconds before the first alaska measurement is taken This can be a maximum of 600 seconds 10 minutes EGR Step5 Duration Enter the time in minutes over which measurements are taken This can be a maximum of 60 minutes ee fe Coa Step 6 Interval Enter the interval time in seconds between 0 New measurements using the left and right arrows Options are 5 10 15 20 30 or 60 seconds Step 7 Press Next O to go to the next parameters screen OR Press Cancel to return to the Functions folder Kinetics Parameters Step 8 Select the measurement Mode using the left and right arrows Delta A change in absorbance over the measurement duration or selected period Final A absorbance at the end of the measurement Ba duration or selected time Slope rate of change of absorbance over the Sony measurement duration or selected period Step 9 Uni
55. ess Cancel to return to the Utilities folder without storing the settings Page 57 68 Miser Sets up Output options P 330 P360 The procedure is as follows Step 1 Output Options Output Device B Cancel Step 2 7 4 Preferences Sets user preferences The procedure is as follows Step 1 Preferences Step 2 Games Auto Standby ee Theme Baseline Compensation Step 3 Step 4 History mu o x maa 7 5 Contrast NanoPhotometer P Class User Manual Select how the data are sent Options are Computer USB Ea and depending on the attached printer module SD Memory Card E Bluetooth Ka or None This can also be set using the Menu key in each application or method If None in the Output Options is selected it is only possible to print with the built in printer if it is connected to the instrument For Computer USB SD Memory Card and Bluetooth the auto print option is always on Press OK O to store the settings and return to the Utilities folder OR Press Cancel to return to the Utilities folder without storing the settings Select Games function This determines whether the games folder is displayed or not Options are yes or no Define the Screen layout Theme of folders Options are either a grid format or a list Select History whether to use previously entered parameters memory function set On or to return to default settings set Off Select whether to use a
56. folder Press 2 to select Protein folder Press 2 to select Protein dye mode Using NanoVolume Applications select the Lid Factor as described in the Average Detection Range Sheet and under 3 2 A minimum of 1 5 ul sample volume for lid 10 is recommended Using Cuvette Applications select Pathlength using the left and right arrows Options are 5 mm or 10 mm Enter the Dilution Factor using the keypad numbers Range 1 00 to 9 999 Use the C button to backspace and clear the last digit entered OR press Menu Options to enter the dilution factor screen Enter the volume of the sample using the keypad numbers Range 0 01 to 9 999 Enter the volume of the diluent using the keypad numbers Range 0 01 to 9 999 Press OK to calculate the dilution factor and return to the Parameters screen OR press Cancel to cancel the selections and return to the Parameters screen Select whether the Dye correction calculation of the dye dependent correction factor is used or not with the left and right arrows The Background correction is always calculated in the Dye methods Select the Protein BSA default Serum Albumin mouse Serum Albumin human IgG Lysozyme Custom or OD 1 If using Custom Protein there are two possibilities to enter the correct factors see also page 18 protein UV method Molarextinctioncoefficient M 14cm 1 Ranges are Wavelength 200 nm to 340 nm Molar extinction coefficient M1 cm 10 000 to 9 999
57. gion of the wavescan kai l it If Off leaves the peak detection as determined on the un zoomed it display 0 20 jei a NG A Sort Peaks by Determines the sequence that peaks are reported OIG Jia proper me by Can be wavelength peak height or peak width Draw Peaks Switches display of peak cursors on and off These show vertical dashed lines displaying the measured peak height and horizontal dashed lines showing the peak width Sample 1 a 450nm Abs 0 164 A Pressing Cancel O ignores the selection pressing OK accepts them Version 1 0 Page 43 68 Miser NanoPhotometer P Class User Manual Add Peak Shortcut button 5 Wavescan User Defined Peak ff 0 30 L Parameters al Data Transfer al Abs 3T a Peak Detection T Delete Peak AA Graph Scale oT Se Sample Number 60 tab a Save Method Output Options 600000000 a nananana ALT nm 4 a 5 nm 4 ob oO 01 3 4 Graph Scale Shortcut button 6 Graph Scale Zoom Mode Version 1 0 Add Peak Shortcut button 5 Adds a user defined peak at the current cursor position The entry is then displayed in inverse colouring to discriminate between user defined peaks and auto detect peaks When the cursor is positioned over the user defined peak a legend User Defined Peak appears at the top of the graph The option then changes to Delete Peak to enable the user to remove the peak Graph Scale Shortcut butt
58. have to be Dye Ext Coefficient Dye Abs Max 300 nm to 950 nm dsDNA Dye Dye Parameters Step 8 If using Custom Dye maximum absorbance wavelength of entered Dye Ext Coefficient 10 000 to 9 999 999 the custom dye dye dependent extinction coefficient and Dye Type Dye Abs Max Ranges are Dye Correction 0 000 to 0 999 Oye Correction 0 000 Results Screen Version 1 0 Page 16 68 Miser NanoPhotometer P Class User Manual dsDNA Dye Results Screen Step9 Apply insert the reference sample Press Blank Key This will be used for all subsequent samples until changed 0 454 Step 10 Apply insert sample and press Sample This measures A280 at the selected wavelengths and displays the results The Concentration sample and dye concentration the FOI and the ratio of Sr A260 A280 and A260 A230 are calculated corrected by the background if selected Step 11 If the absorbance value of the sample is not in the linear range a Warning message will pop up and Instruction O 707 A 0 420 A ADyve 345 0 004 A FO Alexa Fluor 350 will be displayed in the top left corner of the result screen ait Please refer to 3 2 Software instructions important 0 00 pmalful information on page 11 for further information Step 12 Repeat for all samples Step 13 Press Menu Options to display available Options which are described on page 8 Step 14 Press Escape O and confirm with Yes ko to return to the Nucle
59. he instrument will perform a series of self diagnostic checks Please read through this user manual prior to use Please contact your original supplier in the first instance if you experience technical or sample handling difficulties 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 Version 1 0 Page 4 68 Miser NanoPhotometer P Class User Manual 2 INTRODUCTION 2 1 Your spectrophotometer Your spectrophotometer is a simple to use UV Visible instrument with a CCD array detector 1024 pixels It has no moving parts which is the basis of the rapid scanning operating system The user interface is built around folders which are displayed on the main screen when the instrument is switched on Different folders are numbered and opened by using the associated number key on the keypad After switching on the NanoPhotometer a self calibration check is performed and the default main screen NanoPhotometer is offering the Life Science methods such as nucleic acid assays and protein assays using the NanoPhotometer P Class Submicroliter Cell 9 Life Science methods such as nucleic acid assays protein assays and cell density using cuvettes se i Hanovolume Applications St Cuvette NG pplicatons aa General spectroscopic methods _ Functions A Conta
60. hen all standards are measured press OK 9 to accept the calibration and go to the Results screen see below OR press Back Q to cancel selections and return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Press Replicates to display the replicate entry boxes Use C to clear previously stored results before measuring Insert the standard and press Sample to measure the standard and store the result Repeat for all replicates and standards A graph will display the results and the fitted curve as the measurements are input Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Press OK to accept the calibration and go to the Results screen see below OR press Back o to return to the Standards screen Page 33 68 Miser NanoPhotometer P Class User Manual Calibration Screen manual entry Biuret Calibration 0 044 A 0148A Ng EEFT oaaa NPA O4464 na oe 0 542 A ru Ki Dl a Da 04 Off LB LO La 14 K Back o D Results screen 546 nm Concentration Absorbance 0 249 4 Curve Fit Regression Standard Pathlength Sample Pathlength NanoPhotometer P 300 Options select using key
61. ic Acids folder To change parameters print or save methods press the Menu Options button The options menu will be opened For further explanation please see 2 3 Keypad and display on page 6 P 300 and 7 P330 P360 Version 1 0 Page 17 68 Miser NanoPhotometer P Class User Manual 4 2 4 2 Protein Determination 1 General Information Protein determination at 280 nm NanoVolume Applications and Cuvette Applications 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 A280 factor for a particular protein must be determined The protein concentration can be calculated the following way C prot Abs 280 A280 factor lid factor dilution factor With background correction C prot Abs 280 Abs 320 A280 factor lid factor dilution factor This equation can be applied to other proteins if the corresponding factors are known please note that the factor used by the NanoPhotometer P Class is the reciprocal value of the extinction coefficient I g cm from a protein The instrument can determine protein concentration at 280 nm and uses the above equation as default the factors can be changed and the use of background correction at 320 nm is optional The A280 Factor is based on the extinction coefficient of the protein molecular weight
62. id well with a slightly wet fluff free tissue Use water 7096 ethanol or isopropanol Do not use aggressive solvents like strong acids or bases or organic solvents at any time Important Note Residual fluffs must be removed for optimum performance Your cell is ready for the next sample Version 1 0 Page 9 68 Miser NanoPhotometer P Class User Manual Operation Limitations Do not autoclave the unit Do not use an ultrasound bath to clean Do not drop in water or solvent bath The unit is water resistant but not water proof 3 2 Software instructions The NanoVolume Applications and Cuvette Applications are very similar concerning the analysis of dsDNA ssDNA RNA Oligonucleotides protein UV and protein dye analysis This section describes the specific features which have to be considered using the NanoPhotometer P Class Submicroliter Cell For general information please follow the detailed instructions under Nanovolume Applications and Cuvette Applications The procedure is as follows Exemplary Parameter Screen Parameter Screen Step 1 Press 1 to select NanoVolume Applications folder re rameters Step 2 Press 1 to select Nucleic Acids folder OR 2 to select Protein folder Step 3 Select the method you want to use by pressing the corresponding number Dilution Factor Factor Step 4 Select the Lid Factor using the left and right arrows id Sampie volume Pathiength 5 optional 35 5 H 10 optional for P 300 1 3
63. ie 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 The Lowry method is based on the Biuret reaction Under alkaline conditions the divalent copper ion forms a complex with peptide bonds in which it is reduced to a monovalent ion Monovalent copper ion and the radical groups of tyrosine tryptophan and cysteine react with Folin reagent to produce an unstable product that becomes reduced to molyodenum tungsten blue Bound reagent changes colour from yellow to blue This binding is compared with those derived from a standard protein at 750 nm this is usually BSA bovine serum albumin Detailed protocols are supplied with these assay kits and must be closely followed to ensure accurate results are obtained A linear regression analysis of the calibration standard data points is calculated the result together with the correlation coefficient can be
64. incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open Output Options settings possibility to change the Output settings within the method as described in 7 3 Output Options Printer Exit Menu by pressing Escape Q or wait Page 31 68 Miser Biuret Assay NanoPhotometer P Class User Manual The colorimetric Biuret assay is not recommended with the Submicroliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen Biuret Parameters wavelength Pathlength Biuret Parameters Regression Calibration Standards Replicates Standards Screen Biuret Standards Std 1 Std 4 0 800 Std 2 0 400 1 000 Std 3 0 600 1 400 Back Version 1 0 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 Step 12 Step 13 Parameter Screen Press 2 to select Cuvette folder Press 2 to select Protein folder Press 5 to select Biuret mode The default Wavelength setting is 546 nm Enter the number of Standard concentration points 1 9 to be used in the curve using the keypad numbers or left and right arrows Select Pathlength using the left and right arrows Options are 5 mm or 10 mm Units The user can enter a text string up to 8 characters long To
65. ins nine folders that can store user adapted ae methods up to 81 H ser Methods J Utilities 1 5 Instrument set up date time number format Output Options and Baseline Compensation set up The instrument is equipped with a standard USB port The NanoPhotometer P Class Software Package is necessary to connect the NanoPhotometer P Class to a PC The software enables the user to print through the PC directly to the printer that is connected to it Data may be stored as Excel spreadsheet report and or table format EMF graphics file a comma delimited csv data file a tab delimited txt data file or in native NanoPhotometer P Class Software format for later access See also NanoPhotometer P Class Installation and User Manual Alternatively results may be saved on a SD Memory Card or sent to the PC via a Bluetooth accessory these can either be Supplied pre installed or are available as an optional accessory if the need for the use arises after installation of the product A thermal built in printer is available for the instrument this may either be supplied pre installed or is available as an optional accessory if the need for its use arises after installation of the product 2 2 Sample handling tips The NanoPhotometer P Class includes an integrated vortexer P 330 P 360 only to assure a good homogeneity of the sample It is recommended to mix every sample before a measurement Note that the light
66. ions of supply is valid for 12 month for the NanoPhotometer P 300 P 330 and 24 months for the NanoPhotometer P 360 only if the product has been used according to the instructions supplied Implen or your supplier can accept no liability for loss or damage however caused arising from the faulty or incorrect use of this product Version 1 0 Page 64 68 Miser 10 APPENDIX NanoPhotometer P Class User Manual 10 1 Nucleic acid quantification For determination nucleic acid concentration in solution the absorbance at wavelength 260 nm is used The function describing the concentration to absorbance relation is a modification of the Lambert Beer equation C nuc Abs 260 factor nuc lid factor dilution factor With background correction C nuc Abs 260 Abs 320 factor nuc lid factor dilution factor C nuc Abs 260 factor nuc lid factor nucleic acid concentration ng ul absorbance AU of nucleic acids substance specific factor for nucleic acids ng cm ul ds DNA 50 ssDNA 37 RNA 40 Oligo 33 virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid 10 2 Nucleic acid fluorescent dye incorporation To determine the nucleic acid concentration and the dye concentration after probe labelling a modification of the Lambert Beer equation is used Background correction is always calculated possibility to switch the dye correction on and off Calculation of the fluorescence
67. is will be used for all subsequent samples until changed Step 19 Press Replicates Oio display the replicate entry boxes Use C to clear previously stored results before measuring Step 20 Insert the standard and press Sample d to measure the standard and store the result Step 21 Repeat for all replicates and standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 22 Press OK to accept the calibration and go to the Results screen see below OR press Back Q to return to the Standards screen Page 27 68 Miser NanoPhotometer P Class User Manual Calibration Screen manual entry Bradford Calibration 0 058 A 0 196 4 NG D E ee 0 330 4 t 0 463 4 r ZJ a N co i 4 ME g ne 04 D6 OB LO Le Ll4 K Back o D Results screen Bradford 555 nm Concentration Absorbance 0 337 4 Curve Fit Regression Standard Pathlength Sample Pathlength NanoPhotometer P 300 Options select using key pad numbers Parameters Print Graph Edit Sample Pathlensth Sample Number Save Method Printer Settings 0908 S300 NanoPhotometer P 330 P360 Menu select using key pad numbers Parameters Data Transfer Edit Sample Pathlength Sample Mumber
68. length by using keypad numbers or left and right arrows Select the Mode Factor user entered or Standard factor is calculated from a calibration sample using the left and right arrows if Factor is selected Enter the Factor using the keypad numbers Range 0 001 to 9 999 Use the C button to delete the last digit entered if Standard is selected Enter the concentration using keypad numbers Range 0 01 to 9 999 Use the C button to delete the last digit entered Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Menu Options key and then use the left right arrows ug ml ug Ul pmol ul mg ml mmol l umol l g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed Decimal Points DP to be selected from O to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters or Cancel Select the Pathlength using the left and right arrows Options are 0 04 0 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications The Pathlength selection has no influence to the result It is just to document the used pathlength To enter the results screen with the selected parameters press
69. mbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle graph on off Displays the calibration graph cursors give values for last measured sample 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape or wait Menu select using key pad numbers Return to parameters screen Transfer the results via selected Output Options Possibility to edit the sample pathlength Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open Output Options settings possibility to change the Output settings within the method as described in 7 3 Output Options Printer Na NEHA SNA NA 1 2 4 7 Exit Menu by pressing Escape Q or wait Page 34 68 Miser NanoPhotometer P Class User Manual 4 3 Bacterial Cell Culture Measurement OD600 4 3 1 General Information The stage of growth of a bacterial culture needs to be
70. monitored to ensure that the cells are harvested at the optimum point for the greatest density of live cells An exemplary growth curve is given below Cells should be harvested towards the end of the log phase The optical density of the sample indicates when this point has been reached This value varies dependent on the cells being grown Routinely the cells are grown until the absorbance at 600 nm known as OD 600 reaches approximately 0 4 prior to induction or harvesting A linear relationship exists between cell number density and OD 600 up to approx 0 6 stationary Decline Log Phase It is important to note that for turbid samples such as cell cultures the absorbance measured is due to light scattering 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 normalized using calibration curves A calibration curve can be determined by comparing measured OD 600 to expected OD 600 Expected OD 600 is determined by counting cell number using an alternative technique for example microscope slide method and converting to OD 600 using the rule of thumb that 1 OD 600 5 x 108 cells ml for E Coli Additionally your NanoPhotometer P Class is coming wi
71. nd right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open Output Options settings possibility to change the Output settings within the method as described in 7 3 Output Options Printer Exit Menu by pressing Escape 9 or wait Page 51 68 Miser NanoPhotometer P Class User Manual 5 7 Absorbance Ratio This makes simple absorbance ratio measurements on samples measuring the amount of light that has passed through a sample relative to a blank this can be air at two wavelengths The procedure is as follows Parameter Screen Parameter Screen Absorbance Ratio Wavelengths Step 1 Press 3 to select Functions Step 2 Press 7 to select Absorbance Ratio wavelength 1 Wavelength 3 Step3 Enter the first Wavelength by using the keypad numbers or 260 nm the left and right arrows Step4 Enter the second Wavelength as above baka Step5 Select whether a Background correction is applied to both aasa wavelengths 1 and 2 using the left and right arrows Step 6 If background correction is On Enter the third Wavelength from which the background correction will be obtained Step 7 Press Next to enter the next screen OR press Cancel O to return to the Functions folder Background Absorbance Ratio Parameters Step 8 Select the Pathlength using the left and right arrows Options are 0 04 0 1 0 2 1 and 2 mm for NanoVolume Pathlength Factor applications and 5 or 10 mm fo
72. ns will 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 also affects 260 280 readings If a solution is too dilute the readings will be at the instrument s detection limit and results may vary as there is less distinction of the 260 peak and the 280 slope from the Version 1 0 Page 12 68 Miser NanoPhotometer P Class User Manual background absorbance This is one reason why the Abs 260 value should be greater than 0 1 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 A wavelength scan of the nucleic acid 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 can be entered Fluorescent dye incorporation To determine the dye incorporation rate the abs
73. nucleic acid concentration C nuc Abs 260 Abs 320 factor nuc lid factor dilution factor With dye correction C nuc Abs 260 Abs 320 CF dye Abs max dye Abs 320 factor nuc lid factor dilution factor C nuc Abs 260 CF dye Abs max dye factor nuc lid factor nucleic acid concentration ng ul absorbance AU of nucleic acids dye dependent correction factor at 260 nm absorbance at absorption maximum of the dye AU substance specific factor for nucleic acids ng cm ul ds DNA 50 ssDNA 37 RNA 40 Oligo 33 virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid Calculation of the dye concentration C aye ADS max dye Abs 320 lid factor dilution factor aye 108 C dye Abs max dye lid factor E dye dye concentration pmol ul absorbance at absorption maximum of the dye AU virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid dye dependent molar extinction coefficient M1 cmt Calculation of the frequency of incorporation FOI of dye per 1 000 bases FormulafordsDNA FOI 6 49 Abs max dye Abs 320 aye 108 Abs 260 Abs 320 With dye correction FOI 6 49 Abs max dye Abs 320 E aye 108 Abs 260 Abs 320 CF aye ADS max dye Abs 320 Version 1 0 Page 65 68 Miser NanoPhotometer P Class User Manual FormulaforssDNA FOI 8
74. ompanying documents Background colour yellow symbol and outline black Unpacking Positioning and Installation Check the contents of the package against the delivery note If any shortages are discovered inform your supplier immediately 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 Indoor use only Temperature range 5 C to 35 C Note that if you use the instrument in a room subjected to extremes of temperature change during the day it may be necessary to recalibrate by switching off and then on again once thermal equilibrium has been established 2 3 hours Maximum relative humidity of 80 up to 31 C decreasing linearly to 50 at 40 C The instrument must be placed on a stable level bench or table that can take its weight lt 4 5 kg so that air can circulate freely around the instrument This equipment must be connected to the power supply with the power cord supplied It can be used on 90 240 V 50 60 Hz supplies If the instrument has just been unpacked or has been stored in a cold environment it should be allowed to come to thermal equilibrium for 2 3 hours in the laboratory before switching This will prevent calibration failure as a result of internal condensation Switch on the instrument via the keypad after it has been plugged in T
75. on 6 This enables the user to set up a defined graph by defining the limits in either or both of the x and y axes Zoom mode This sets up the operation of the Zoom keys up and down arrows The x amp y axis expands the display around the cursor measurement point whilst the other options select the absorbance or wavelength axes respectively With x or y axis limits set to on zooming out will only be permitted to the set limits x y axis limits Setting x or y axis limits to On activates the start and finish points of the desired graph to user defined specific wavelengths and or absorbance values Pressing Cancel Q ignores the selection pressing OK 9 accepts it and displays the required graph Page 44 68 Miser NanoPhotometer P Class User Manual 5 4 Kinetics Simple kinetics studies where the change in absorbance needs to be followed as a function of time at a fixed wavelength can be readily performed Reagent test kits are routinely used for the enzymatic determination of compounds in food beverage and clinical laboratories by measuring NAD NADH conversion at 340 nm The change in absorbance over a specified time period can be used to provide useful information when an appropriate factor defined in the reagent kit protocol is applied Reaction rate and enzyme activity can be calculated if the factor used takes account of the absorbance difference per unit time as opposed to the absorbance diff
76. on the Background correction Step 6 Select the Protein BSA default Serum Albumin mouse Serum Albumin human IgG Lysozyme Custom or OD 1 Background Protein UY Parameters Step 7 If using Custom Protein there are two possibilities to enter the correct factors Molarextinctioncoefficient M1 cm Protein wavelength Ranges are Wavelength 200 nm to 340 nm 7 Molar extinction coefficient M1 cm 10 000 to 9 999 999 tele ei a ell Molecular weight 0 001 to 9 999 999 47730 Molecular weight 69323 3985 Extinctioncoefficient I g cm Ranges are Protein Wavelengih Wavelength 200 nm to 340 nm ERETTE Extinction coefficient I g cm 0 001 to 9 999 Step 8 Select the Units of measurement using the left and right arrows Options mg ml ug ml ng ul and ug ul Step 9 Press OK to enter the Results screen OR Cancel to return to the Protein folder Protein UY Parameters Ext coefficient li g cm 0 650 bib Ok oS Cancel Version 1 0 Page 19 68 Miser NanoPhotometer P Class User Manual Results Screen Results Screen Step 10 Apply insert the reference sample Press Blank Key This will Protein LI Y be used for all subsequent samples until changed Step 11 Apply insert sample and press Sample This measures at both 260 and 280 nm wavelengths and displays the result Protein concentration is calculated corrected by background wavelength value if selected Step
77. orbance reading at the wavelength reported for maximum absorbance of the fluorescence dye is used The corresponding extinction coefficient of the dye is used in the Lambert Beer Law to determine the dye concentration c A e d Comparing these values with the DNA concentration gives a dye incorporation rate For further details please refer to 10 2 Nucleic acid fluorescent dye incorporation Use of Background Correction 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 If it is used there will be different results from those when unused because Abs 320 is subtracted from Abs 260 and Abs 280 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 If your laboratory has not used background correction before set this option to OFF The use of background correction can remove variability due to handling effects of low volume disposable cells Spectral scan of nucleic acid Pure Nucleic Acid Poly dAdT Wave 260 0 Abs 0 567 Prai Wave 280 0 Abs 0 409 Absorbance A 0 1 0 0 T 210 0 260 0 310 0 36
78. ored in the folder User Methods o MA ee E ai Folder names can be renamed locked unlocked and saved to the SD memory card using the Options Menu Menu Options select using key pad numbers 1 Folder Names gt Lock Folder E Unlock Folder Ch S0 Memory Card Rename Folder Names 1 Press 1 to select Folder Names 2 Select the method to be renamed using the left and right arrows 3 Enter the new name 4 Press OK to save the new name OR Cancel to return to the User Methods folder Lock Method 1 Press 2 to select Lock Folder 2 Select the method to be locked using the left and right arrows 3 Select a pass code using the keypad numbers or left and right arrows 4 Press OK to lock the method OR Cancel Q to return to the User Methods folder Unlock Method 1 Press 3 to select Unlock Folder Select the method to be unlocked using the left and right arrows Enter the pass code using the keypad numbers or left and right arrows Press OK to unlock the method OR Cancel O to return to the User Methods folder a Io SD Memory Card Individual or all methods can be copied on the SD Memory Card and can be restored back into the same instrument at a later date For further details please refer to the NanoPhotometer P Class Accessory manual 1 Press 4 to select SD Memory Card 2 Four options are available Backup folder generates a copy of an individual folder on the SD Memory Card
79. otides cccceeeeeeeeeeeeeeeeeseeeeeeeeeseeeeeeeeeeeneneees 12 4 1 1 Generalii onma paaa NN AAO lan Agana 12 4 1 2 Analysis of dsDNA ssDNA and RNA 0 cee a 14 4 1 3 Analysis of Oligonucleotides 1 reen 15 4 1 4 Dye incorporation for dsDNA ssDNA RNA and Oligonucleotides ccccecsseeeeeeeeeeeeees 16 4 2 Protein Determination NAAALAALA 18 4 2 1 Cae eane dan 1e AEA NA AA AA A E EE 18 Gaz PEON UV MEMO aaa Nena a E E kA haaa 19 4 2 3 Protein UV Dye Method 1110171 21 124 BONO A iain secassegeescaaemas ekaeen ced dectestaationd aca thitens 4 ccjavirnonenecyeidirnneneeceansaueos te netien 23 BUO TAS SV aaa AA AA NN NAN NGA AA KANG 32 4 3 Bacterial Cell Culture Measurement OD600 ccccceseeeseeeeeeenseeeeeeasseeesceasseeseeenseeeseoenseeesooees 35 4 3 1 General TOMA ON AA Karan aa aa naa 35 4 3 2 Analysis of Bacterial Growth aa 36 PUNG NONG EK AGA AA AA 3 5 1 Single Wavelength ADS and f aa AA 38 az CONCERT ANON oaa OARE EEE EEA ae SA Ei 40 Do Wave SCa AA AA PAA AA 41 DA FIN Lo AA AA AA AA AA 41 55 SAI AN CUVE AA AAP RANI NAG RAR PAA PAKA MR 41 5 6 Multiple VV AV CIOINQ UN saa AA AA 41 Dal PADSOMD AN Ce PANIC AAP AA AA AA AA AA AA 41 USER METHOD Ss andaunsstcocdhensseseccecasecesit soe heseescioabe E E E 41 OPETE AA AA AA AA AA AA AA AA AA AA AA AA AA 41 Cl Dale ARNG N AA AA AA AA AA AA 41 Te AA AA AA AA AA AA AA 41 LS Qutput
80. pad numbers Parameters Print Graph Edit Sample Pathlensth Sample Number Save Method Printer Settings 0908 S300 NanoPhotometer P 330 P360 Menu select using key pad numbers Parameters Osta Transfer Edit Sample Pathlength Sample Mumber Save Method Output Options LL o O o o O Version 1 0 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad Step 23 The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range is 0 001 to 9 999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Step 24 Press OK gt to accept the calibration and go to the Results screen See below OR press Back Q to return to the Standards screen Results screen Step 25 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step26 Insert the sample and press Sample QD The concentration of the sample is taken and displayed Step27 Repeat for all samples Step 28 Press Menu Options to display available Options which are described below Step 29 Press Escape and confirm with Yes 9 iv return to the Protein folder Query needs confirmation to avoid unintended escaping the application Options select using key pad nu
81. plays the results The sample concentration and the ratio of A260 A280 and A260 A230 are calculated corrected by the background wavelength value if selected If the absorbance value of the sample is not in the linear range a Warning message will pop up and Instruction will be displayed in the top left corner of the result screen Please refer to 3 2 Software instructions important information on page 11 for further information Repeat for all samples Press Menu Options to display available Options which are described on page 8 Press Escape O and confirm with Yes to return to the Nucleic Acids folder To change parameters print or save methods press the Menu Options button The options menu will be opened For further explanation please see 2 3 Keypad and display on page 6 P 300 and 7 P330 P 360 Version 1 0 Page 15 68 Miser NanoPhotometer P Class User Manual 4 1 4 Dye incorporation for dsDNA ssDNA RNA and Oligonucleotides The dye incorporation methods are similar to the dsDNA ssDNA RNA and Oligonucleotide methods This section describes the specific features concerning the dye incorporation For general information please follow the detailed instructions under Analysis of dsDNA ssDNA and RNA and Oligonucleotides To determine the dye incorporation rate the absorbance reading at the wavelength reported for maximum absorbance of the fluorescence dye is used For further details please refer to 10 2 N
82. ples until changed Step 16 Insert sample and press Sample NW Step 17 Repeat for all samples The absorbance at the selected wavelengths is measured and the ratio between wavelengths 1 and 2 is calculated both corrected by the background wavelength value if this was selected Step 18 Press Menu Options to display available options which are described below Step 19 Press Escape O and confirm with Yes to return to the Functions folder Query needs confirmation to avoid unintended escaping the application Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options Printer settings 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open Output Options Printer settings possibility to change the Output Options Printer settings within the method as described in 7 3 Output Options Printer Exit Menu by pressing Escape Q or wait Page 53 68 Miser NanoPhotometer P Class User Manual 6 USER METHODS These folders are the storage locations for any user modified Applications Methods that are saved in the Options Menu They are accessible from the home folders page The folder enables the user to quickly select any frequently used Methods Up to 9 Methods may be st
83. pment EN61326 1 2006 Electromagnetic compatibility generic emission standard electrical equipment for measurement control amp laboratory use For further information including unpacking positioning and installation of the products please refer to the user manual Signed Dated October 1 2011 as Dr Thomas Sahiri Managing Director Implen GmbH Version 1 0 Page 2 68 Miser NanoPhotometer P Class User Manual TABLE OF CONTENTS 1 2 10 BINA ET NOTE sate catcher NAAAGNAS 4 NTR ODI NON APA AA AA AA tnd gundnctmnedentaded AA AA E 5 2 1 Your spectrophotometer ssssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn AA AA AA 5 22 Sampie handling UPS AA AA Aia AA PAA PBA 5 2 3 Keypad and display for NanoPhotometer P 300 ssansnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnna 6 2 4 Keypad and display for NanoPhotometer P 330 P 360 nnasnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnna 7 2 5 MNU ORSON S eis sei ce et ste cco ta ns vasa AERE EA annniioe ssa tense snanatanatacaedeninsatuuedcnexneeeeavacdebanatsusees 8 THE NANOPHOTOMETER P CLASS SUBMICROLITER CELL ma a BANANA NAGA 9 amp l PEGCIRICAN INS FUCTION S 727x ceencancsnccesanstetacantesmecacatnanccesiaanedeeseuntedasnansmecenssiatwessasdvavwestasaneesssassaneecsenns 9 3 2 SONWare IMSIMUIGIION S ABA AABANG AA 10 NANOVOLUME APPLICATIONS AND CUVETTE APPLICATIONS nsa GALA 12 4 1 Characterization of DNA RNA and Oligonucle
84. press the down arrow and enter name 9 Open Output Options settings possibility to change the Output settings within the method as described in 7 3 Output Options Printer Exit Menu by pressing Escape O or wait Page 25 68 Miser NanoPhotometer P Class User Manual Bradford Assay The colorimetric Bradford assay is not recommended with the Submicroliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen Bradford Parameters wavelength Pathlength Units Scone Bradford Parameters Calibration 4 Standards Replicates Version 1 0 Parameter Screen Step 1 Press 2 to select Cuvette folder Step 2 Press 2 to select Protein folder Step 3 Press 3 to select Bradford mode Step 4 The default Wavelength setting is 595 nm Step 5 Enter the number of Standard concentration points 1 9 to be used in the curve using the keypad numbers or left and right arrows Step 6 Select Pathlength using the left and right arrows Options are 5 or 10 mm Step 7 Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Menu Options key and then use the left right arrows ug ml ug ul pmol ul mg ml mmol l umol 1 g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed Decimal Points DP to be selected from O to 2
85. quent samples until changed Step 26 Insert the sample and press Sample O The concentration of the sample is taken and displayed Step 27 Repeat for all samples Step 28 Press Menu Options to display available options which are described below Step 29 Press Escape and confirm with Yes to return to the Protein folder Query needs confirmation to avoid unintended escaping the application Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle graph on off Displays the calibration graph cursors give values for last measured sample 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9
86. r Dye methods 220 nm to 750 nm with cursors denoting 230 260 P 330 and P 360 only 280 and 320 nm Nucleic Acid methods and 260 280 and 320 nm Protein methods Arrow Keys Alphanumeric Keys Version 1 0 Page 7 68 Miser NanoPhotometer P Class User Manual 2 5 Menu Qptions select using key pad numbers Options for P 300 Ca After each measurement the following Options are possible Gb Parameters l 1 Return to parameter screen Pi 2 Print the results via selected method Graph 3 Toggle graph on off Graph shows a wavescan plot across the GP Print Data Only range 220 nm to 400 nm for Dye methods 220 nm to 750 nm with cursors denoting 230 260 280 and 320 nm 4 Toggle on off the graph in the print out or saved file E Sample Number 7 Define the sample number you wish to start from E save Method 8 Save the parameters as a method P Printer Settings 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape OR wait Menu options for P 330 P 360 After each measurement the following options are possible in the Gb Parameters Menu 3 Data Transfer Return to parameter screen Transfer the results via selected Output Option Toggle on off the graph in the print out or saved file Define the sample number you wish to start from Save the parameters as a method
87. r cuvette applications The Pathlength selection has no influence to the result It is just to document the used pathlength mi Ga Step9 Dilution Factor known Enter a Dilution factor by using the 1 000 keypad numbers within the range 1 00 9 999 Units way rol lt i gt OK E Back Step 10 Calculate Dilution Factor Press the Menu Options key sa ih Maa Enter the Volume of the sample range 0 01 9 999 Dilution Factor j using the keypad numbers Enter the volume of Diluent range 0 01 9 999 by using the keypad numbers ERE Step 11 Press OK Od to calculate the dilution factor and return to the Parameters screen OR press Cancel to cancel Diluent selections 0 000 Step 12 Select units of measurement using left and right arrows Options are ug ml ng ul ug ul Step 13 Enter the factor using the keypad numbers Range 0 001 to 9 999 Step 14 Press OK Sr to enter the results screen OR Cancel to return to the Functions folder E Fa 2g Fe amp Cancel a Fa EEE a aa aan ni Hi ESSE SEEMED a E e Version 1 0 Page 52 68 Miser NanoPhotometer P Class User Manual Results Screen Absorbance Ratio 12 9 pa mi Menu select using key pad numbers Gb Parameters t3 Data Transfer F Sample Number Sawe Method E Output Options Version 1 0 Results Screen Step 15 Insert the reference sample Press Blank key This will be used for all subsequent sam
88. rdless of how many decimal points are selected So 98768 2 will display as 98768 even with 1 decimal point selected Press OK chosen parameters or Cancel Q to store the Select the type of Curve Fit using the left and right arrows Options straight line regression a zero regression this forces the straight line through the origin interpolated or cubic spline Select the Calibration mode either Standards measure prepared standards or Manual keypad data entry or New Standards previous values are blanked new standard can be measured if standards selected Select the number of standards to be measured and averaged at each standard concentration point Can be Off 1 2 or 3 Step 10 Press Next to enter the Standards screen OR Press Cancel O to cancel selections and return to the Functions folder Page 47 68 Miser NanoPhotometer P Class User Manual Standard Screen Standard Curve Standards Standard Curve Calibration A 10 0 00154 nas ik 20 0 0 049 4 Pa 30 0 0 082 4 a 0 10 rab 40 0 0 115 A Fa 0 1478 Fa ms 0 1795 0 05 na ta a 20 30 40 50 Bo S m Calibration Screen replicates on Standard Curve Calibration Replicates oos24 amp gas Ha it 20 ad 4g 50 B0 Version 1 0 Standards screen Step 11 Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard
89. ress Menu Options to display available options which are described below Step 13 Press Escape and confirm with Yes gt to return to the Functions folder Query needs confirmation to avoid unintended escaping the application folder Page 38 68 Miser NanoPhotometer P Class User Manual NanoPhotometer P 300 Options select using key pad numbers OY 83009 Options select using key pad numbers 1 Return to parameters screen Parameters Baca 2 Print result via selected method et 3 Toggle between Absorbance and T mode a A Print graph greyed out if no data are available Pe ier 7 Sample number add a prefix to the sample number and reset Sample Number Save Method Printer Settings NanoPhotometer P 330 P 360 Menu select using key pad numbers Ei OO8 Parameters Data Transfer Abs ET Print Graph Sample Number Save Method Output Options Version 1 0 the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options 3
90. rned E save Method to this position the legend User Defined Peak is displayed at Output Options the top of the scan and this option changes to Delete Peak 6 Displays Graph Scale Parameter Screen See description below 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in Favourites Methods 1 9 press the down arrow and enter name 9 Open Output Options Printer settings possibility to change the Output settings within the method as described in 7 3 Output Options Printer Exit Menu Options by pressing Escape or wait Peak Detection Shortcut button 4 Peak Detection Shortcut button 4 Peak Detection Auto Detect Peaks Turns on and off the automatic peak detection The following options determine how peaks are detected Minimum Peak Height Minimum height the peak has to be above the higher of the two adjacent minima for the peak to be detected Minimum Peak Width Minimum width of the peak as determined by the difference in wavelength between the highest of the two adjacent minima and the opposing intersection of that higher Min Pk Width Draw Peak ni i minimum level and the peak profile See the screen displayed nm o Br Ne below Hika OF E Cancel Peak Detect on Zoom Determines whether peaks are re assessed fi and tabulated when the user zooms into a re
91. rner of the result screen Please refer to 3 2 Software instructions important information on page 11 for further information Repeat for all samples Press Menu Options to display available options which are described on page 8 Press Escape and confirm with Yes O to return to the Nucleic Acids folder To change parameters print or save methods press the Menu Options button The options menu will be opened For further explanation please see 2 3 Keypad and display on 6 P 300 and 7 P330 P 360 Version 1 0 Page 14 68 Miser NanoPhotometer P Class User Manual 4 1 3 Analysis of Oligonucleotides The procedure is as follows Parameter Screen NanoVolume Applications Step 1 Oligo Parameters Step 2 Step 3 Lid Factor Units Step 4 Dilution Factor Step 8 Background Cuvette Applications Oligo Parameters Step 6 Pathlength Units Step 7 Step 8 Dilution Factor Step 9 Background gt OK Step 10 Results Screen Step 11 Step 12 Step 13 Step 14 Step 15 Step 16 Parameter Screen Press 1 for NanoVolume OR 2 for Cuvette folder Press 1 to select Nucleic Acids folder Press 4 to select Oligo mode Using the NanoVolume Applications select the Lid Factor as described in the Average Detection Range Sheet and under 3 2 Using Cuvette Applications select Pathlength using the left and right arrows Options are 5 mm or 10 mm Enter the Dilution Factor using the keypad numbers R
92. rt the reference sample Press Blank Key This will be used for all subsequent samples until changed Step 15 Apply insert sample and press Sample This measures at 260nm 280nm 320nm and the dye specific wavelength and displays the result Protein concentration corrected by background wavelength value if selected dye concentration and degree of labelling is calculated Step 16 If the absorbance value of the sample is not in the linear range a Warning message will pop up and Instruction will be displayed in the top left corner of the result screen Please refer 3 2 Software instructions important information on page 11 for further information Step 17 Repeat for all samples Step 18 Press Menu Options to display available Options which are described on page 8 Step 19 Press Escape O and confirm with Yes to return to the Protein folder To change parameters print or save methods press the Menu Options button The options menu will be opened For further explanation please see 2 3 Keypad and display on 6 P 300 and 7 P330 P 360 Version 1 0 Page 22 68 Miser NanoPhotometer P Class User Manual 4 2 4 BCA Assay The colorimetric BCA assay is not recommended with the Submicroliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen Parameter Screen BCA Parameters Step 1 Press 2 to select Cuvette folder Step 2 Press 2 to select Protein folder Pathlength Step3 Press
93. s select using key pad numbers Parameters Print Graph Edit Sample Pathlength Sample Number Save Method Printer Settings 609 6000 NanoPhotometer P 330 P360 Menu select using key pad numbers Sb Parameters 3 D ata Transfer Ch Edit Sample Pathlength F Sample Humber E Save Method Output Options Version 1 0 NanoPhotometer P Class User Manual Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle graph on off Displays the calibration graph cursors give values for last measured sample 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape O or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left a
94. s to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Insert the standard use C to clear previously stored results before measuring Press Sample to measure the standard and store the result Repeat for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading When all standards are measured press OK 9 to accept the calibration and go to the Results screen see below OR press Back to cancel selections and return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Press Replicates to display the replicate entry boxes Use C to clear previously stored results before measuring Insert the standard and press Sample to measure the standard and store the result Repeat for all replicates and standards A graph will display the results and the fitted curve as the measurements are input Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Press OK to accept the calibration and go to the Results screen see below
95. samples or solvents Changing cell holder or removal for cleaning This can be removed by undoing the appropriate screws on the bottom of the instrument The symbol 8 on the product or on the documents accompanying the product indicates that this appliance may not he treated as household waste Instead it shall be handed over to the applicable collection point for the recycling of electrical and electronic equipment Disposal must be carried out in accordance with local environmental regulations for waste disposal Version 1 0 Page 61 68 Miser NanoPhotometer P Class User Manual Error messages and Trouble shooting 8 6 Error messages Error text in display Explanation Calibration failure UV on Reference channel Cell holder obstructed No light on Reference channel Calibration problem UV IR on Reference channel Waiting for software update Keyboard Calibration problem possible lamp failure Submicroliter cell or cuvette in the cell holder by switching on the instrument Instrument was too cold Submicroliter cell or cuvette in the cell holder by switching on the instrument Submicroliter cell or cuvette in the cell holder by switching on the instrument Cell holder has been removed from the instrument and placed back in the wrong position Low light levels The instrument has been turned on and off too fast Insufficient provision of electricity Power supply does not deliver 18V
96. sequent samples until changed Step 15 Insert the standard use C to clear previously stored results before measuring Press Sample D io measure the standard and store the result Step 16 Repeat for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 17 When all standards are measured press ox to accept the calibration and go to the Results screen See below OR press Back Bio cancel selections and return to the Standards screen Calibration Screen replicates on Step 18 This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 19 Press Next to display the replicate entry boxes Use C to clear previously stored results before measuring Step 20 Insert the standard and press Sample to measure the standard and store the result Step 21 Repeat for all replicates and standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 22 Press OK to accept the calibration and go to the Results screen See below OR press Back to return to the Stan
97. solution of dsDNA in a 10 mm pathlength cell with an optical density of 1 0 has a concentration of 50 ug ml ssDNA of 37 ug ml or 40 ug ml in the case of RNA Oligonucleotides 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 Please refer to 10 1 Nucleic acid quantification for further details The instrument uses factors 50 37 40 and 33 as default settings for dsDNA ssDNA RNA and Oligonucleotides respectively and compensation factors for dilution and use of cells which do not have 10 mm pathlength Dilution factor and cell pathlength can be entered Nucleic Acid Purity Checks Nucleic acids extracted from cells are 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 an indication however and not a definitive assessment Pure DNA and RNA preparations have expected ratios of 1 8 and 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 in 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 variatio
98. td 6 0 600 1 400 i il Dika Mest B Back Version 1 0 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 Step 12 Step 13 Parameter Screen Press 2 to select Cuvette folder Press 2 to select Protein folder Press 4 to select Lowry mode The default Wavelength setting is 750 nm Enter the number of Standard concentration points 1 9 to be used in the curve using the keypad numbers or left and right arrows Select Pathlength using the left and right arrows Options are 5 or 10 mm Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Menu Options key and then use the left right arrows ug ml ug ul pmol ul mg ml mmol l umol 1 g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed Decimal Points DP to be selected from O to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected So 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters OR Cancel Q Press Next Oio enter the next screen Select the Calibration mode either Standards measure prepared standards Manual keypad data entry or New Standards previous values are blanked new standard can be measured if Standards selected Select th
99. th a correction factor of 1 as default To compare OD values between different spectrophotometer you have to determine the constant deviation between the Absorbance values for the same sample within those instruments and use this factor within the setting correction factor of your NanoPhotometer P Class Software The use of 10 mm 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 The Submicroliter Cell is not recommended for optical density measurements of cell culture solutions Version 1 0 Page 35 68 Miser NanoPhotometer P Class User Manual 4 3 2 Analysis of Bacterial Growth The procedure is as follows Parameter Screen OD 600 Parameters Step 1 Step 2 wavelength Step 3 B00 nm Step 4 Correction 1 000 Step 5 OD 600 Parameters Step 6 Wavelength Factor coo rm Step 7 Correction Multiplier Units Step 8 Results Screen Step 9 tamad S50 nm 10 Absorbance cells ml Step 11 0 010 A Step 12 Step 13 1000 000 Parameter Screen Press 2 to select Cuvette Applications Press 3 to select OD 600 Select the Wavelength Default value is 600 nm Range is 200 nm to 950 nm Enter the Correction factor to compensate for different optical configurations between this and other instruments Default value is 1 Select the Units Options
100. tput Options Printer settings 3 Print all the data 4 Set the to position starting point for the slope and dA calculation at the current cursor position Value is retained for subsequent samples 5 Set the tn position finishing point for the slope and dA calculation at the current cursor position Value is retained for subsequent samples 6 Toggle the calculated slope line on and off Note if any data points enclosed by to and tn are beyond the range of the instrument gt 2 5 A or lt 0 3 A then this option is greyed out 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open Output Options Printer settings possibility to change the Output Printer settings within the method as described in 7 3 Output Options Printer Exit Wenu Options by pressing Escape or wait Page 46 68 Miser 5 5 Standard Curve NanoPhotometer P Class User Manual The construction of a multi point calibration curve from standards of known concentration to quantify unknown samples is a fundamental use of a spectrophotometer this instrument has the advantage of being able to store this curve as a method using up to 9 standards To include a zero concentration standard include this in the number of standards to be entered and enter 0 0
101. ts The user can enter a text string up to 8 characters long To access a list of pre defined units press the Menu Options key and then use the left right arrows lt i gt DK Back ug ml ug Ul pmol ul mg ml mmol l umol l g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed Decimal Points DP to be selected from O to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK Sd to store the chosen parameters OR Cancel Q Step 10 Set the Factor by which the result is multiplied to give the amount in the chosen range using the left and right arrows Range of 0 01 to 9 999 Step 11 Select the Pathlength using the left and right arrows Options are 0 04 0 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications The Pathlength selection has no influence to the result It is just to document the used pathlength aes Sa a nan eanan aan a ee PAP RISES ESS SSSI SDSS SSS kaa Step 12 Press Next to enter the Results screen OR press Cancel O to return to the Parameters screen Version 1 0 Page 45 68 Miser NanoPhotometer P Class User Manual Results Screen 0 20 fy BAe oa 0 10 I 0 05 l l l l l l Ni l l l l l l l l
102. u NANOPHOTOMETER Alphanumeric Keys Ton Arrow Keys Escape Cancel Back ample Sample Enter selection OK Blank Reference Key Action On Off Key Turns the instrument on off Use the four arrow keys to navigate around the display and select the required setting from the active highlighted option View menu for that application mode Some of these are common to all View Menu applications and described on page 8 Menu unique to an application are described in the relevant section of the NanoPhotometer P Class User Manual Use these to enter parameters and to write text descriptions where appropriate or required Use repeated key presses to cycle through lower case number and upper case Leave for 1 second before entering next character Use C button to backspace and 1 to enter a space Escape Cancel Back Escape from a selection and return to the previous folder Cancel a selection Stop making measurements O Blank Reference Set reference to 0 000 A or 100 T on a reference solution at the current wavelength in the mode selected When in scan mode does a reference scan Sample Enter Selection OK gt Enter or confirm a selection Take a measurement Print Prints the results shown on the screen on the built in printer if a built in printer is P 330 and P 360 only connected to the NanoPhotometer Toggle graph on off The graph shows a wavescan plot across the range 220 nm to Graph Data 400 nm fo
103. ucleic acid fluorescent dye incorporation The procedure is as follows Parameter Screen NanoVolume Applications Parameter Screen dsDNA Dye Parameters Step 1 Press 1 for NanoVolume OR 2 for Cuvette folder Step 2 Press 1 to select Nucleic Acids folder Lid Factor lass Step 3 Press 5 6 7 or 8 to select one of the dye incorporation methods Step 4 Using the NanoVolume Applications select the Lid Factor as Dilution Factor Factor described in the Average Detection Range Sheet and ee under 3 2 Using Cuvette Applications select Pathlength using the left and right arrows Options are 5 mm or 10 mm Dye Correction Dye Type Step5 Select Dilution Factor Units and Factor as described under 4 1 2 Step 6 Select whether the Dye correction calculation of the dye e o ce dependent correction factor is used or not with the left and right arrows The Background correction is always calculated S in the Dye methods Cuvette Applications Step 7 Select the appropriate Dye Type 10 different AlexaFluors 4 dsDNA Dye Parameters Cy Dyes 6 Oyster Dyes and Texas Red are programmed with their corresponding maximum absorbance wavelength dye Pathlength Units dependent correction factor at 260 nm and dye dependent extinction coefficient For further details please refer to 10 2 Nucleic acid fluorescent dye incorporation Dilution Factor Factor Dye Correction Dye Type dye dependent correction factor at 260 nm
104. umber to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape or wait Menu select using key pad numbers Return to parameters screen Transfer the results via selected Output Options Possibility to edit the sample pathlength Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open Output Options settings possibility to change the Output settings within the method as described in 7 3 Output Options Printer Db a N3 1 2 4 T Exit Menu by pressing Escape or wait Page 28 68 Miser Lowry Assay NanoPhotometer P Class User Manual The colorimetric Lowry assay is not recommended with the Submicroliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen Lowry Parameters wavelength Pathlength Lowry Parameters Regression Calibration Standards Replicates Cancel Standards Screen Lowry Standards Std 1 Std 4 0 800 Std_ 2 Std 5 0 400 1 000 Std 3 S

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