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USER MANUAL

1. ssessssssseeseeeeeeeenen nnne 59 r dE iuum TENENEL G M 60 EC m G 60 TO oU rc 60 8 uiu did rise S 61 9 1 Maintenance Iree TecnnologQgy ioesu conia ee oon env conn o au aus np sus Do Sn nC Sana c reD ni eEonsSS Ue Usa aaia EUER aaa 61 02 Lamp Fa SN A IM MI uate en teeter tens centennial ei ieee a ee ieee MEER Uu 61 99 Cos o lel pili E E eee e mee nee p nen aCe ne ea mene ame Sn aoe e marae 61 94 Exchange ofihe ganer mE ewe ecco cease ie eee neem en 61 8 5 Cleaning and general care of the instrument cccceeeseeeeeeeenseeeseeenseeeseoenseeeseoeneeesseoeeneeessooeees 62 00 dit cl Pee E 63 Version 2 1 NanoPhotometer P Class User Manual Page 3 7O Qu NanoPhotometer P Class User Manual o7 TOUDI Aloe ro pfo NER 64 9 AO C Shots 0 64 10 SPECIFICATION AND WARRANTY sascseccsscusinsnceewunbnssuaceuacdunsasd aninsusananseususucnasuasaususbesandduavaussncasdansdnssvecessnecevessnsesssssceuinniess 65 TI SOLVENT COMPATIBILITY escavus sivasu t i8g RN OF EREERENE NER SAAE 66 MEME d pp dere S
2. SX 67 12 1 NUGIEIC acid guante ANO eme n 67 12 2 Nucleic acid fluorescent dye INCOrPOratiON ccccceecseeeeeeeeseeeeeeeeeseeeeeceenseeeeeoeenseeeeeoensneeessoeenseees 67 12 3 Protein CUANUINC ATOR ssis M 69 12 4 Protein fluorescent dye incorporation cccsecseeeeeeeesseeeeeeeenseeeeeeeenseeeeeeoenseeeeeoonseeessooesseesseooenseees 69 Version 2 1 Page 4 70 Qu 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 N Caution refer to accompanying documents Background colour yellow symbol and outline black Unpacking Positioning and Installation e Check the contents of the package against the delivery note If any shortages are discovered inform your supplier immediately e Inspect the instrument for any signs of damage caused in transit If any damage is discovered inform your supplier immediately e 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
3. QS NanoPhotometer P Class User Manual P 300 P 330 P 360 Version 2 1 P 360 IMPLEN telephone 49 89 726 3718 O Fax 49 89 726 3718 54 Email info implen de www implen de NanoPhotometer P Class User Manual 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 EN61010 2 101 2002 Particular requirements for IVD medical equipment EN61
4. 50 100 and 250 dependent on the used dilution lid E dye dye dependent molar extinction coefficient M cm e Calculation of the frequency of incorporation FOI of dye per 1 000 bases Formula for dsDNA FOI 6 49 Abs max aye Abs 320 aye 10 Abs 260 Abs 320 With dye correction FOI 6 49 Abs max aye Abs 320 dye 10 Abs 260 Abs 320 CF aye Abs max aye Abs 320 Version 2 1 Page 67 70 Qu NanoPhotometer P Class User Manual Formula for ssDNA FOI 8 77 Abs max dye Abs 320 aye 10 Abs 260 Abs 320 With dye correction FOI 8 77 Abs max aye Abs 320 dye 105 Abs 260 Abs 320 CF aye Abs max aye Abs 320 Formula for RNA FOI 8 11 Abs max dye Abs 320 aye 10 Abs 260 Abs 320 With dye correction FOI 8 11 Abs max aye Abs 320 dye 10 Abs 260 Abs 320 CF aye Abs max aye Abs 320 Formula for Oligonucleotides FOI 9 83 Abs max dye Abs 320 aye 10 Abs 260 Abs 320 With dye correction FOI 9 83 Abs max aye Abs 320 aye 10 Abs 260 Abs 320 CF aye AbS max aye Abs 320 Abs max dye absorbance at absorption maximum of the dye AU dye dye dependent extinction coefficient M cm The following dye types and parameters are pre programmed in the NanoPhotometer P Class nm coeffi
5. 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 M 1 cm t 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 12 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 Coomassie Brilliant Blue to an unknown protein and comparing this binding to that of different known concentrations of a standard protein at 595 nm this is usually BSA bovine serum albumin The Biuret method depends on
6. Important Note Residual fluffs must be removed for optimum performance Your cell is ready for the next sample Version 2 1 Page 10 70 Qu 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 ASOMA S Parameters 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 Step 4 Select the Lid Factor using the left and right arrows Dilution Factor Factor Pathlength Dilution 5 optional 35 5 2mm 15 optional for P 300 1 3pgl 1mm 110 Background qe Ba E Cancel 03 2u 02mm 150 100 optional 0 3 2 ul 1 100 250 optional 0 3 2 yl 1 250 Step 5 Select subsequent parameters and
7. Page 50 70 Qu 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 Step 2 Press 6 to select Multi Wavelength Step 3 Select the number of Wavelengths pt Step 4 Press OK to enter the next screen Step 5 Enter the first Wavelength using either the number keys or the left and right arrows Step 6 Enter the second Wavelength as above and repeat for the number of wavelengths selected up to 5 Step 7 Press OK o to enter the results screen OR press Cancel to return to the Functions folder Pathlength Z2 OK amp Cancel Results Screen Step 8 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Multi wavelength Step 9 Insert sample and press Sample m 300 nm o Step 10 Repeat for all samples A scan plot covering the range of gem Td wavelengths selected with cursors at the relevant T wavelengths and a table of values is displayed E 500 nm o Step 11 Press Menu Options to display available options which SES are described below 300 400 nn B ES HN i PRI it a INU Ty du 300 400 coo Step 12 Press Escape E and confirm with Yes o to return to the Functions folder Quer
8. 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 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 pmol l g l mg l ug l U I 96 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 O7 to 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 Qo to enter the Standards screen OR press Cancel to cancel selections and re
9. This will be used for all subsequent samples until changed Step 18 Press Replicates Qo to display the replicate entry boxes Use C to clear previously stored results before measuring Step 19 Insert the standard and press Sample Qo to measure the standard and store the result Step 20 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 21 Press OK M to accept the calibration and go to the Results screen see below OR press Back to return to the Standards screen Page 28 70 Version 2 1 Qu NanoPhotometer P Class User Manual Calibration Screen manual entry Bradford Calibration 0 200 0 058 A E 0 400 D 186 amp EPA D E d 0 600 0 330 A vv o 4 0 800 0 463 4 1 400 0 7134 Bs og oa Ln DE LH Ln Ld LM E OK E Back Results screen Bradford 835 nm Absorbance 0 337 amp Curve Fit Regression Standard Pathlength Sample Pathlength NanoPhotometer P 300 Options select using key pad numbers Parameters Frint Graph Edit Sample Pathlength Sample Humber Save Method Printer Settings NanoPhotometer P 330 P360 Menu select using key pad numbers Farameters ata Transfer Edit Sample Farhlenath Sample Humb
10. see below OR press Back to return to the Standards screen Results screen Step 24 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 25Insert the sample and press Sample Q5 The concentration of the sample is taken and displayed Step 26 Repeat for all samples Step 27 Press Menu Options to display available options which are described below Step 28 Press Escape L and confirm with Yes M 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 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 L or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use t
11. used for all subsequent samples until changed Step 15 Insert sample and press Sample Step 16 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 17 Press Menu Options to display available options which are described below Step 18 Press Escape 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 T 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 LI or wait Page 54 70 Qu 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
12. 3 2 NanoPhotometer P 330 P 360 The NanoPhotometer P Class offers a selection of data output accessories Please proceed to Folder 5 Utilities press 3 Output Options to set the favoured reporting settings Data output to Built in printer Printing on the built in printer is possible independent from the Output Options setting with the Print key on the keypad Pressing the Print key prints the results shown on the result screen on the built in printer If no built in printer is connected to the NanoPhotometer P Class the Print key results in a warning message If None in the Output Options is selected it is only possible to print with the built in printer or save the data on a connected USB flash drive No data None transfer to a PC is possible To transfer the data on the result screen to a PC press 9 for Output Options change the Output Options to the device of choice Press OK Qo to store the settings and return to the result screen Press Menu or directly 2 for Data Transfer to transfer the data to the PC All further measurements are transferred and saved automatically lt gt OK 2 Cancel Direct Data output to USB flash drive If a USB flash drive is connected to the USB flash drive port right side of the NanoPhotometer all measured data are automatically saved on the USB flash drive independent from the Output Options settings Please exit the method first before disconnecting the USB flash drive Otherwise the
13. 5 Manually adds a peak position to the peak table in the results screen at the position set by the cursor If the cursor is returned to this position the legend User Defined Peak is displayed at the top of the scan and this option changes to Delete Peak 6 Displays Graph Scale Parameter Screen See description below 4 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 L or wait Peak Detection Shortcut button 4 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 minimum level and the peak profile See the screen displayed below Peak Detect on Zoom Determines whether peaks are re assessed and tabulated when the user zooms into a region of the wavescan If Off leaves t
14. 600 2a Lo 0 800 pa mn E O88 LU lZ LH LB E Back Bradford Calibration 0 200 0 058 A E 0 400 0 185 4 Fd 0 6 2 0 600 0 330 4 rg 0 800 0 462 4 T y 1 000 D 582 4 Fd 1 400 D 718 4 fo Bs 4 UN 0 842 A j na D BB UB LD le LH LB o Calibration Screen replicates on Bradford Calibration Replicates Replicates 0 718 A POM 0 7154 v 0 2198 4 D 4 0 718 A a ma x NS L4 O86 0E lO Ld L4 Calibration Screen replicates off Step 13 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 14 Insert the standard use C to clear previously stored results before measuring Press Next to measure the standard and store the result Step 15 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 16 When all standards are measured press OK Qo 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 Step 17 This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key
15. Abs 280 Abs 320 aye With dye correction degree of labelling Abs max aye Abs 320 prot Abs 280 Abs 320 Abs max aye Abs 320 CF aye aye Abs max dye absorbance at absorption maximum of the dye AU E prot protein dependent molar extinction coefficient M cmt CF aye dye dependent correction factor at 280 nm to be delivered from dye supplier dye dye dependent molar extinction coefficient M1 cmt The following dye types and parameters are pre programmed in the NanoPhotometer P Class Dyes nm coefficient aye factor 280 nm CFpye Cy3 es X 25000 005 PadficBue 416 34600 020 PE 58 20000 048 TeasRed 595 8000 048 n all formulas the molar dye dependent and the molar protein dependent extinction coefficient is used Version 2 1 Page 70 70
16. Insert the sample and press Sample Qo 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 14 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 15 Press Menu Options to display available options which are described below Step 16 Press Escape E and confirm with Yes Qo 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 Output 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 2 5 A or 0 3 A then this opt
17. 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 Qo to 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 Qo 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 to return to the Parameter screen Page 33 7O Qu NanoPhotometer P Class User Manual Calibration Screen replicates off Biuret Calibration Step 13 pub 0 400 is per LD n fia n O6 ME LU lZ LH E Back Biuret Calibration Step 15 0 200 0 5 a 0 800 E A Step 16 Dl ma O4 O86 0E LO Le LU o Calibration Screen replicates on Biuret Calibration Step 17 gt Replicates 1 0 2349 A tos 0 345 A i D u posee Step 18 hi 4 0 800 0 3439 A ba a Step 19 nl n D4 LB 1 8
18. Switch the device Off and disconnect the power supply reconnect it after about 5 minutes Firmware update version 2 4 2 recommended Thermal paper printer prints a plenty of Thermal Printer paper was probably put the wrong way check paper normal sound no error message correct position of the printer paper Thermal printer automatic roller stops Please contact the Implen support team Please contact the Implen Support Team supportGimplen 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 2 1 Page 64 70 Qu NanoPhotometer P Class User Manual 10 SPECIFICATION AND WARRANTY Technical Specifications Spectrophotometer Wavelength range 190 1 100 nm Wavelength scan range 200 950nm System start up time Less than 5 seconds no warm up necessary Measure time for full scan 3 5 seconds range lt 0 596 at 220 nm using Nal and 340 nm using NaNO2 0 003 A 0 to 0 5 A 0 007 A 0 5 1 0 A 260 nm Noe 0002Amsat0AG260nm 0 005 A pk to pjat OAG 260nm 15 mm centre height z height outside dimension 12 5 mm x 12 5 mm NanoVolume application Detection range dsDNA 2 ng ul to 18 750 ng ul BSA 0 08 mg ml to 543 mg ml Absorbance range 0 01 1 5 10 mm equivalent 0 05 375 0 3 yi Path lengths 0 04 mm 0 1 mm 0 2 mm 1 mm and 2 mm Virtual dilution facto
19. Up to 9 Methods may be stored in the folder User Methods Methods 1 Methods 2 eV e 55 c Le iul N 2 d ethadz i NES PE EE dim Tod y Methods S JF DU S erhad AER EIS minm ta fm Fam oc mg Mes T Res i 1 Methods 3 E JU Miss 5 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 d Folder Hames E Lock Folder E Unlock Folder c SD Memon Card Rename Folder Names 1 Press 1to select Folder Names 2 Select the method to be renamed using the left and right arrows 3 Enter the new name 4 Press OK o 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 o to lock the method OR Cancel to return to the User Methods folder Unlock Method 1 Press 3 to select Unlock Folder 2 Select the method to be unlocked using the left and right arrows 3 Enter the pass code using the keypad numbers or left and right arrows 4 Press OK o to unlock the method OR Cancel Q to return to the User Methods folder 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
20. 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 Q to return to the Standards screen Page 34 7O Qu NanoPhotometer P Class User Manual Calibration Screen manual entry Biuret Calibration 0 200 D 044 amp A 0 400 0 14684 0 5 if 0 600 0 249 4 e f 0 3 0 800 0 349 4 1 000 0 446 4 1 400 0 542 4 Wn ral Ui I4 16 LH LU L8 LH E OK E Back Results screen B46 nm Absorbance 0 249 4 Curve Fit Regression Standard Pathlength Sample Pathlength NanoPhotometer P 300 Options select using key pad numbers Parameters Frint Graph Edit Sample Pathlength Sample Humber Save Method Printer Settings NanoPhotometer P 330 P360 Menu select using key pad numbers Farameters ata Transfer Edit Sample Farhlenath Sample Humber Save Method Output Options uli e o e e Version 2 1 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad Step 22 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 23 Press
21. 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 10 cells ml for E Coli Additionally your NanoPhotometer P Class is coming with 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 2 1 Page 36 7O Qu NanoPhotometer P Class User Manual 4 3 2 Analysis of Bacterial Growth The procedure is as follows Parameter Screen OD 600 Parameters wavelength B00 nm Correction 71 000 OD 600 Parameters w
22. cells Spectral scan of nucleic acid Pure Nucleic Acid Poly dAdT o co e u Wave 260 0 Abs 0 567 o o o a Wave 280 0 Abs 0 409 Absorbance A o w 210 0 260 0 310 0 360 0 410 0 Wavelength nm Note e absorbance maximum near 260 nm and absorbance minimum near 230 nm e flat peak near 260 nm and steep slope at 280 nm e very little absorbance at 320 nm 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 2 1 Page 14 70 4 1 2 Analysis of dsDNA ssDNA and RNA The procedure is as follows Parameter Screen NanoVolume Applications dsl HA Parameters Lid Factar Units Dilution Factor Factor Cuvette Applications ERE UT Parameters Pathlength Units Dilution Factor Factor Results Screen Ame ooa Amo onea Ame oona A2b56075780 Concentration a j 5 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 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 Applicatio
23. 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 5096 at 40 C e 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 e The equipment should be positioned such that in the event of an emergency the mains plug can be easily located and removed e 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 e 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 e Switch on the instrument via the keypad after it has been plugged in The instrument will perform a series of self diagnostic checks e Please read through this user manual prior to use e 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 2 1 Page 5 7
24. data can be lost On the right corner of the display is the status of the USB flash drive shown USB flash drive is connected Data are written to the USB flash drive Open file on the USB flash drive Please exiting the method first before disconnecting the USB flash drive Version 2 1 Page 58 70 Qu NanoPhotometer P Class User Manual Direct Data output to PC Connection between NanoPhotometer P Class and PC is possible with a USB cable connection Select how the data are send Options are Computer USB or None Press OK Output Options MO to store the settings and return to the Utilities folder OR press Cancel Q to return to the Utilities folder without storing the settings If Computer USB is selected all measured data are sent automatically to the PC The file is closed and saved once the method is left by pressing Cancel o or parameters are changed in the parameter settings ox 7 3 3 Loading changing the printer paper 1 Lift off the cover 3 Paper gripped Lock the platen and turn the wheel to feed the paper 2 Feed in the paper 4 Cover replaced Normally the paper is automatically gripped Sometimes it helps if the platen lock is released Version 2 1 Page 59 70 Qu 7 4 Preferences Sets user preferences The procedure is as follows Auto or Baseline EE CE Preferences History E Cancel 7 5 Contrast Step 1 Step 2 Step 3 Step 4 S
25. is used or not with the left and right arrows It is recommended to switch on the Background correction 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 Molar extinction coefficient M cm Ranges are Wavelength 200 nm to 340 nm Molar extinction coefficient M cm t 10 000 to 9 999 999 Molecular weight 0 001 to 9 999 999 Extinction coefficient l g cm Ranges are Wavelength 200 nm to 340 nm Extinction coefficient l g cm 0 001 to 9 999 Select the Units of measurement using the left and right arrows Options mg ml ug ml ng yl and ug pl Press OK to enter the Results screen OR Cancel to return to the Protein folder Page 20 7O Qu Results Screen NanoPhotometer P Class User Manual Step 10 Step 11 ABO Sample D 388 A E A280 Result Step 12 D 012 A 131 A320 0 003 4 Units Step 13 Lay mi Step 14 Step 15 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 v This measures at both 260 and 280 nm wavelengths and displays the result Protein concentration is calculated corrected by backgroun
26. printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape L or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options 3 Toggle between Absorbance and T mode 4 Print graph greyed out if no data are available T 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 E or wait Page 40 70 Qu NanoPhotometer P Class User Manual 5 2 Concentration 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 Parameter Screen Concentration Parameters Step 1 Press 3 to select Functions Step 2 Press 2 to select Concentration Step 3 Set Wavelength by using keypad numbers
27. 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 2 1 Page 11 70 Qu 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 Message Answer ves Answer No Ud Concentration too Tow EE DR NK 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 Lid10 Concentration too low NENNEN NMNMMAMANM 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 Concentration too low Please change to lid 10 apply a minimum of ipl of No changes Do you want to change the lid factor sample and press sample automatic change of lid factor lid 50 to lid 10 in the software for calculation Concentration too high Dilute sample or change to lid 100 Concentration too low Please change to lid 50 and press sample No changes Do you want to change the lid factor automatic change of lid factor
28. the Year Step 5 Enter the Minute Seconds are zeroed when OK is pressed Step6 Press OK Qo to store the settings and return to the Utilities folder OR press Cancel to return to the Utilities OK Camel folder without storing the time 7 2 Regional Sets Number Format The procedure is as follows Regional Step 1 Set the Number Format decimal point style Options are ai or pon Humber Format 4 335 3 Ld Step 2 Press OK Qo to store the settings and return to the Utilities folder OR press Cancel to return to the Utilities folder without storing the settings Z2 DE E Cancel 4 3 Output Options Printer 7 3 1 NanoPhotometer P 300 The NanoPhotometer P 300 offers a selection of data output accessories Please proceed to Folder 5 Utilities press 3 Printer to set the requested reporting settings Data output to Built in printer Step 1 Select whether Auto print is on or off using the left and right arrows When auto print is set as on the results are automatically printed Auto Print ES after a measurement is taken When the setting is off printing has to be initiated manually This can also be set using the Options key Base in each application or method The default is OFF Step 2 Select printer module Built in Press OK M to store the settings and return to the Utilities folder OR Press Cancel to return to the Utilities folder without storing the settings ae OK amp Cance
29. 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 2 1 Page 38 70 Qu NanoPhotometer P Class User Manual 5 1 Single Wavelength Abs and 96T This makes simple absorbance A and 96 transmission T measurements on samples measuring the amount of light that has passed through a sample relative to a reference this can be air The procedure is as follows Parameter Screen Single Wavelength Parameters Results Screen Single Wavelength 150 100 mn Cancel Single Wavelength Parameter Screen Step 1 Press 3 to select Functions Step 2 Press 1 to select Single Wavelength Step 3 Set Wavelength by using keypad numbers or left and right arrows Step 4 Select the Mode Absorbance or Transmission using the left and right arrows Step 5 To enter the results screen with the selected parameters press OK Qo OR cancel the selections and return to the Functions folder by pressing Cancel LI Results Screen Step 6 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 7 Insert sample and press Sample Q5 Step 8 Repeat for all samples Step9 The result at the
30. 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 supportGimplen 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 2 1 Page 63 7O Qu NanoPhotometer P Class User Manual 8 7 Trouble shooting Symptom Instrument failing start up calibration Check that the correct power supply 18 V 1 2 A is 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
31. 0 Qu 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 choice of HanoPhotometer M i number Life Science methods such as nucleic acid assays and protein assays using the NanoPhotometer P Class Submicroliter Cell Life Science methods such as nucleic acid assays protein assays and cell density using cuvettes i M anov alume pplications 6 Lunette M pplications Em General spectroscopic methods Functions Contains nine folders that can store user adapted UT methods up to 81 LI zer Methods c 5 Instrument set up date time number format Utilities Output Options and Baseline Compensation set up The instrument is equipped with a standard USB port back and the P330 P360 also with a USB flash drive port right side The NanoPhotometer P Class Software Package is necessary to connect the NanoPhotometer P Class to a PC or to read the
32. 0 P360 Version 2 1 Page 18 70 Qu NanoPhotometer P Class User Manual 4 2 Protein Determination 4 2 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 l 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 molar extinction coefficient M 1 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
33. 13 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 14 Insert the standard use C to clear previously stored results before measuring Press Sample to measure the standard and store the result Step 15 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 16 When all standards are measured press OK O7 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 Step 17 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 18 Press Next Qo to display the replicate entry boxes Use C to clear previously stored results before measuring Step 19 Insert the standard and press Sample Qo to measure the standard and store the result Step 20 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 cle
34. 326 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 Dr Thomas Sahiri Managing Director Implen GmbH Version 2 1 Page 2 70 Qu TABLE OF CONTENTS L ESSENTIAL SAFETY NOTES siipien A A ESEUN ON riw NEN du ra EA DH E NE 5 Unpacking Positioning and Installation lesse eieeeee eei eeeee ene e eene n enn n hannah nana nnn 5 2 INTRODUCTION e 6 ZA Your sSDeciropHhiotomie l6 ecoecantic con sex ce o eame n conne dU nn a aE EE 6 22 Sample handling tips usce ones mr eet renee air ud sas du exam tweet cee rav DU eeu Ramp Cu cu Hato dre Edd deinde 6 2 3 Keypad and display for NanoPhotometer x e n 7 2 4 Keypad and display for NanoPhotometer P 330 P 360 8 25 TSU ODEHOPNS uerunt o oai curd ast ad ben iicr eos escent enews damit ide Cd sns imr vivmd cern Pa eau tiet du qu ERARE 9 3 THE NANOPHOTOMETERS P CLASS SUBMICROLITER CELL 11eeeeee eere eene eene neun nuu u uuu u uuu u uuu u uuu u uuu u uuu u uuu us 10 MEN I dni igbegiteec 10 3 2 Softwar
35. 60 absorbance AU of nucleic acids factor nuc substance specific factor for nucleic acids ng cm ul ds DNA 50 ssDNA 37 RNA 40 Oligo 33 lid factor virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid 12 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 e Calculation of the fluorescence 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 aye AbS max aye Abs 320 factor nuc lid factor dilution factor C nuc nucleic acid concentration ng pl Abs 260 absorbance AU of nucleic acids CF aye dye dependent correction factor at 260 nm Abs max dye absorbance at absorption maximum of the dye AU factor nuc substance specific factor for nucleic acids ng cm ul ds DNA 50 ssDNA 37 RNA 40 Oligo 33 lid factor virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid e Calculation of the dye concentration C dye ADS max aye Abs 320 lid factor dilution factor aye 10 C dye dye concentration pmol l Abs max dye absorbance at absorption maximum of the dye AU lid factor virtual dilution factor 5 10
36. Analysis of Bacterial Growth ccccccccccsssseeecceeeseeeecceeesececeseauaeceeesseuseeeesseauaeeeessaaeeeessseaes 37 5 diee lm Y 38 5 1 Single Wavelength ADS and f iouiscsasescopussassucccsesuss novus ss ases Ec oa re vba VO cus EFE ZEE au EUM KcCEaE Sg FN nnna 39 292 CODCOnIl QUOI uon cnini cutus ua vn I sam xS E RII US Ne EEUU NIME E dcs SES Ca P UPCIAD CE SUEEEIE RUN 41 D39 cie coast 43 S4 KIN OS e 46 S MEE Cl i CONO a E a renee ae oer eee eter eter 48 5 6 Multiple Wavelength sanssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn UIN SN nnmnnn nnmnnn nnmnnn 51 S7 ADSON FAN san E E E 53 6 USER METHODS cea aces eines E EEE EE E 55 7 UTILES raana EAE EE E E EEEE E O U 56 EE i e Mui s eee ec say eect eeetie aeteee eee eee eeegeteeneeae S lt netecenieecmsexsueeeueensues 57 E unit 57 7 3 Output Options Printer 57 7 3 1 NanoPhotometer giis TEE 57 7 3 2 NanoPhotometer P 330 P 360 ccccsccssssesesescsvececececesescevevecacaceceucevevevavavevereesevevavavveneneneevens 58 7 3 3 Loading changing the printer paper
37. E E ET d Step 6 Step 7 Step 8 Step 9 Calibration Standards Replicates at Mest amp Cancel Standards Screen BCA Standards Std 1 Std 4 Std 5 0 400 1 000 Std amp 0 600 1 400 O n Version 2 1 Step 10 Press Next Step 12 Press Next Parameter Screen Press 2 to select Cuvette folder Press 2 to select Protein folder Press 2 to select BCA mode The default Wavelength setting is 562 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 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 l 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 Q Press Next to 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 b
38. LO l2 LM Seu Step 20 Step 21 Version 2 1 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 When all standards are measured press OK Qo 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 Qo 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
39. Lid Factor as described in the Average Detection Range Sheet and under 3 2 A minimum of 1 5 pl sample volume for lid 10 is recommended Step 5 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 Step 6 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 Step 7 Select the Protein BSA default Serum Albumin mouse Serum Albumin human IgG Lysozyme Custom or OD 1 Step 8 If using Custom Protein there are two possibilities to enter the correct factors see also page 18 protein UV method Molar extinction coefficient M cm Ranges are Wavelength 200 nm to 340 nm Molar extinction coefficient M cm t 10 000 to 9 999 999 Molecular weight 0 001 to 9 999 999 Extinction coefficient l g cm Ranges are Wavelength 200 nm to 340 nm Extinction coefficient l g cm 0 001 to 9 999 Step 9 Select the Units of measur
40. NTENANCE 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 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 re
41. OK Qo to accept the calibration and go to the Results screen see below OR press Back to return to the Standards screen Results screen Step 24 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 25 Insert the sample and press Sample Q5 The concentration of the sample is taken and displayed Step 26 Repeat for all samples Step 27 Press Menu Options to display available Options which are described below Step 28 Press Escape L and confirm with Yes M 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 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 L or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options T Sample number add a prefix to the sample number and reset the in
42. Step 14 Apply insert the reference sample Press Blank Key This will be used for all subsequent samples until changed Step 15 Apply insert sample and press Sample VV 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 E and confirm with Yes Qo 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 2 1 Page 23 7O Qu 7 3 4 BCA Assay NanoPhotometer P Class User Manual The colorimetric BCA assay is not recommended with the Submicroliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen BCA Parameters Step 1 Step 2 Pathlength Step 3 Step 4 Step 5 562 mm Units BCA Parameters DP iu Mr T E
43. Volume 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 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 800 under 3 2 Step 5 Select Dilution Factor Units and Factor as described under Dye Correction Dye Type 4 1 2 Step 6 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 Step 7 Select the appropriate Dye Type 10 different Alexa Fluors Cuvette Applications 4 Cy Dyes 6 Oyster Dyes and Texas Red are programmed dsDNA Dye Parameters with their corresponding maximum absorbance wavelength dye dependent correction factor at 260 nm and dye dependent extinction coefficient For further details please refer to 12 2 Nucleic acid fluorescent dye incorporation Dilution Factor Factor Dye Correction Dye Type dsDNA Dye Dye Parameters Step 8 If using Custom Dye maximum absorbance wavelength of dye dependent correction factor at 260 nm have to be entered Dye Ext Coefficient Dye Abs Max 300 nm to 950 nm Dye Ext Coefficient 10 000 to 9 999 999 the custom dye dye dependent extinction coe
44. absorption spectrum can be obtained from your instrument enabling simple identification of peak height and position The procedure is as follows Parameter Screen Wravescan Parameters Pathlength Start wavelength End Wavelength Hode Z2 OK amp Cancel Measurement Screen wW avescan Sample A 450nmi Results Screen Ww auezcarn 0 30 L 0 25 ji 0 20 m n 15 I A 0 10 I il l faa 0 05 j v I Lo a 1 Em Abs 0 154 4 Version 2 1 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 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 To enter the measurements screen with the selected parameters press OK 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 for all subsequent samples until changed Insert sample and press Sample 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
45. alculated 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 L and confirm with Yes M 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 2 1 Page 16 70 Qu 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 12 2 Nucleic acid fluorescent dye incorporation The procedure is as follows Parameter Screen Nano
46. anoPhotometer 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 yl 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 1 3 Output Options Printer 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 Protein determination at 280 nm Submicroliter Cell Cuvette Christian Warburg Protein Dye Protein determination at 280 nm and dye Submicroliter Cell Cuvette incorporation 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 The NanoVolume Applications folder and the Cuvette Applications folder contain different sub folders Nucleic Acids Protein and OD 600 Cell Density Contents
47. ar the previous reading Step 21Press OK to accept the calibration and go to the Results screen see below OR press Back to return to the Standards screen Page 25 70 Calibration Screen manual entry BCA Calibration 0 200 0 051 A 0 400 0 171 A Ki 0 800 M Pd 1 000 Pi 0 627 4 Dd ur 1E D4 D6 1 8 LO l2 LM E OK E Back Results screen FB nm Units pua ml Absorbance _ _ 0 255 4 Curve Fit Regression Standard Pathlength Sample Pathlength 10 mm NanoPhotometer P 300 Options select using key pad numbers Parameters Print Graph Edit Sample Pathlength Sample Muriber Save Method Printer Settings NanoPhotometer P 330 P360 Menu select using key pad numbers Farameters Osta Transfer Edit Sample Farhlenath Sample Humber Save Method Output Options a e o e e Version 2 1 NanoPhotometer P Class User Manual Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad Step 22 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 23 Press OK Qo to accept the calibration and go to the Results screen
48. ave 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 L or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options 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 m or wait Page 29 7O Qu NanoPhotometer P Class User Manual 7 3 6 Lowry Assay The colorimetric Lowry assay is not recommended with the Submicroliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen Lowry Parameters Units gt Mest E Cancel Lowry Parameters Regression Calibration Standards Heplicatez D Ment E Cancel Standards Screen Lowry Standards Version 2 1 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 Step 12 Parameter Screen
49. avelength Factor Correction Multiplier 71 000 4 x 1000 000 Ld Units cells ml a OK E Cancel Results Screen wavelength S50 nm Absorbance cells mi 0 0704 1000 000 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 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 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 Qo 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 Q5 The wavelength absorbance and OD60O0 value
50. c Keys Arrow Keys Sample Enter selection OK 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 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 Stop making measurements 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 Enter or confirm a selection Take a measurement Prints the results shown on the screen on the built in printer if a built in printer is connected to the NanoPhotometer Toggle graph on off The graph shows a wavescan plot across the range 220 nm to 400 nm for Dye methods 220 nm to 750 nm with cursors denoting 230 260 280 and 320 nm Nucleic Acid methods and 260 280 and 320 nm Protein methods Page 8 70 Qu 2 5 Menu Options select using key pad numbers Options for P 300 Param
51. cape L 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 4 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 L 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 LI or wait Page 42 70 Qu 5 3 Wavescan NanoPhotometer P Class User Manual An
52. centration Step9 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 10 Insert sample and press Sample W Version 2 1 Page 41 70 Qu NanoPhotometer P Class User Manual Results Screen if using standard mode Concentration i Run Standard Concentration wavelength zb nm Factor wal mil S571 NanoPhotometer P 300 Options select using key pad numbers Parameters Frint Graph Run Standard Sample Humber Save Method Frinter Settings NanoPhotometer P 330 P 360 Menu select using key pad numbers 1 Farameterz E Osta Transfer Run Standard es Sample M umber Sawe Method Output Options Version 2 1 Results Screen if using standard mode Step 11 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 12 Press Sample Qo to display the Run Standard screen Step 13 Run the standard by pressing Run o OR Press Cancel E to return to the measure screen Step 14Insert the sample and press Sample The concentration of the sample is displayed Results shown as indicate the concentration is out of range Step 15 Repeat for all samples Step 16 Press Menu Options to display available options which are described below Step 17 Press Es
53. cient Eaye factor 260 nm CFpye AlexaFluor647 65o 239000 O 000 Alexa Fiuor660 660 107000 000 Alexa Fuor680 680 164000 O00 Oyters0 8505 7800 gt 029 Oyser 56 660 20000 004 n all formulas the molar dye dependent extinction coefficient is used Version 2 1 Page 68 7O Qu NanoPhotometer P Class User Manual 12 3 Protein quantification 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 t cm prot or 1 extinction coefficient I g cm lid factor virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid A280 factors pre programmed Serumi Serum albumin BSA albumin IgG Lysozyme human mouse A280 factor 0 71 0 38 Molecular Weight g mol 69 323 4 68 692 5 69 365 7 150 000 14 300 Molar extinction coefficient M t cm t 47 790 43 780 39 310 210 000 37 500 12 4 Protein fluorescent dye incorporation To determine the protein concentration a
54. crementing 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 m or wait Page 35 70 Qu 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 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 O 4 prior to induction or harvesting A linear relationship exists between cell number density and OD 600 up to approx 0 6 otationary 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
55. d in 7 3 Output Options Printer Exit Menu by pressing Escape Q OR wait Page 9 70 Qu NanoPhotometer P Class User Manual 3 THE NANOPHOTOMETER P CLASS SUBMICROLITER CELL With its innovative optical pathway the cell is designed for optimum measurement results with submicroliter samples ranging from O 3 pl 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 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
56. d 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 L 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 page 6 P 300 and 7 P330 P 360 Version 2 1 Page 21 70 Qu NanoPhotometer P Class User Manual 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 ive Correction de OF E Cancel Cuvette Applications Protein Oye Parameters Pathlength Protein Dilution Factor Units Dye Correction qe OK e Cancel Protein Ove Dye Parameters Dyve Type Oye Abs Max Oye Ext Coefficient SOOO Oye Correction Version 2 1 Parameter Screen Step 1 Press 1 for NanoVolume OR 2 for Cuvette folder Step 2 Press 2 to select Protein folder Step 3 Press 2 to select Protein dye mode Step 4 Using NanoVolume Applications select the
57. e instructions E ES 11 4 NANOVOLUME APPLICATIONS AND CUVETTE APPLICATIONS creen e eene e enne ener nnne nanus a nnns a anna nana nnus 13 4 1 Characterization of DNA RNA and Oligonucleotides eeeeeeeeeeeess 13 4 1 1 General InformiallODk siscsccctsecatnsniondestadancasnaaencnvaad snes tneeavnanadtnddanenpndsidaaaeeivadaniensntdaadantanondsitanamedars 13 4 1 2 Analysis of dsDNA ssDNA and RNA cccccceeecccceeeeeeceeeeeeeeaeceeceeeeeesaeeeeesseaeceeseaeeeesaaeeeeeas 15 4 1 3 A Analysis of Oligonucleotides cece ccccccceeeeeeeeceeeeeeeeeseaeeeceeessaaeeeeeesseaseeeeesseseeeeessaeeeeeeesaess 16 4 1 4 Dye incorporation for dsDNA ssDNA RNA and Oligonucleotides 17 42 Prot in Determination RE 19 4 2 1 General Information eesssseesssssessssesesseeesenn nennen EEEE AEE EE 19 4 2 2 Protein UV Method eee 20 4 2 3 Protein UV Dye Method P m 22 T324 BOCAASSIy oca ee E eee ene 24 1 35 3 6216 0 6 ASSAY MT an eee nn eee nee ee ee eee eee ee ene J 27 7 3 6 LOWY PSSA RR Or E E E E E 30 fid sisi 33 4 3 Bacterial Cell Culture Measurement OD600 1111 Lee ee Leee ee Lleeern eene nennen anna nana mua 36 4 3 1 General denne Tm 36 4 3 2
58. e 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 to enter the Standards screen OR press Cancel to cancel selections and return to the Protein folder Standards Screen Step 11Enter 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 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 to return to the Parameter screen Page 24 70 Qu Calibration Screen replicates off BCA Calibration 25 MEER 0 400 0 600 Leg 0 800 Lo 1 400 Te ni L4 DLE QE LO le LU ECA Calibration 0 6 i 0 200 0 051 A 0 400 D1r0A ms Dr 0 600 0 2884 ay s 0 800 0 403 4 ri 4 4 1 000 516 A 1 400 0 626 4 ty 3r EI E o L4 QE 0E LU le LH lt gt OF E Back Calibration Screen replicates on BCA Calibration 0 6 w H 0 626 4 x 2 0 626 A ns En 0 4 1 400 0 676 A 0 2 Pi E Di qa Dn BB BH LU Le LH O w Version 2 1 NanoPhotometer P Class User Manual Calibration Screen replicates off Step
59. e 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 E C l Back 9 Escape from a selection and return to the previous folder Cancel a selection scape vance Bae 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 9 Enter or confirm a selection Take a measurement Alphanumeric keys Version 2 1 Page 7 70 Qu 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 ON Off Key Escape Cancel Back Blank Reference Key On Off Key Arrow Keys View Menu Alphanumeric Keys Escape Cancel Back Q Blank Reference Sample Enter Selection OK Print P 330 and P 360 only Graph Data P 330 and P 360 only Version 2 1 IMPLEN Print with Built in Printer Toggle Graph Data View Menu NANOPHOTOMETER Alphanumeri
60. elected 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 Step 7 Press Next to enter the next screen Step8 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 Step9 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 10 Press Next o to enter the Standards screen OR press Cancel to cancel selections and return to the Protein folder Standards Screen Step 11Enter 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 12 Press Next M 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 to return to the Parameter screen Page 27 70 Qu NanoPhotometer P Class User Manual Calibration Screen replicates off Bradford Calibration dh ae 0 400 0
61. ement using the left and right arrows Options mg ml ug ml ng ul and pg pl Step 10 Enter the protein dependent extinction coefficient Range is 10 000 to 9 999 999 Step 11 Press OK o 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 12 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 22 70 Qu NanoPhotometer P Class User Manual Results Screen cay Ay Protein Oye Sample P LLLLL fr Protein Concentration ALD ve 3465 464 ua ml ive Concentration 0 00 pmol ul Degree of Labelling 0 00 Results Screen
62. er Save Method Output Options uli e o e e Version 2 1 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad Step 22 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 23 Press OK Qo to accept the calibration and go to the Results screen see below OR press Back to return to the Standards screen Results screen Step 24 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 25Insert the sample and press Sample Q The concentration of the sample is taken and displayed Step 26 Repeat for all samples Step 27 Press Menu Options to display available Options which are described below Step 28 Press Escape L and confirm with Yes Qo 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 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 S
63. eters Frint Graph Print O ata Only Sample Muriber Save Method Printer Settings O90 S500 Menu options for P 330 P 360 Osta Transfer a Parameters e Output Data Only o v Sample li urmber Save Methad Output Options Version 2 1 NanoPhotometer P Class User Manual After each measurement the following Options are possible 1 2 3 4 T 8 9 Return to parameter screen Print the results via selected method Toggle graph on off Graph shows a wavescan plot across the range 220 nm to 400 nm for Dye methods 220 nm to 750 nm with cursors denoting 230 260 280 and 320 nm 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 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 L OR wait After each measurement the following options are possible in the Menu 1 2 4 1 8 9 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 Open Output Options settings possibility to change the Output Options settings within the method as describe
64. fficient and Dye Type Dye Abs Max Ranges are Dye Correction 0 000 to 0 999 Oye Correction 0 000 Version 2 1 Page 17 70 Qu NanoPhotometer P Class User Manual Results Screen Results Screen o CEA dsDNA Dye Step9 Apply insert the reference sample Press Blank Key This will be used for all subsequent samples until changed Step 10 Apply insert sample and press Sample This measures at the selected wavelengths and displays the results The sample and dye concentration the FOI and the ratio of Concentration bas 00 srt A260 A280 and A260 A230 are calculated corrected by 245 00 pa mil i J the background if selected gU Step 11 If the absorbance value of the sample is not in the linear ADyet345 0 range a Warning message will pop up and Instruction a will be displayed in the top left corner of the result screen AZ60 A280 158 Dye Concentration Please refer to 3 2 Software instructions important AZ60 A230 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 L 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 P33
65. 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 variations will at 260 nm Thus different instruments of the same and different types may give slightly Version 2 1 Page 13 7O Qu NanoPhotometer P Class User Manual 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 background absorbance This is one reason why the Abs 260 value by using the submicroliter cell NanoVolume applications should stay between than 0 01 and 1 50 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 t
66. he 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 E or wait Page 26 7O Qu NanoPhotometer P Class User Manual 7 3 5 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 wef avelength Pathlength 535 nm Units La mil 4 Ment E Cancel Bradford Parameters Calibration Standards Replicates Version 2 1 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 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 l pmol ul mg ml mmol l pmol l g l mg l ug l U I 96 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 s
67. he nucleic acid is particularly useful for RNA samples The instrument can display 260 280 and 260 230 ratios Fluorescent dye incorporation To determine the dye incorporation rate 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 Comparing these values with the DNA concentration gives a dye incorporation rate For further details please refer to 12 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
68. he peak detection as determined on the un zoomed display Sort Peaks by Determines the sequence that peaks are reported 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 Pressing Cancel L ignores the selection pressing OK Qo accepts them Page 44 70 Qu NanoPhotometer P Class User Manual Add Peak Shortcut button 5 w auezcar User Defined Peak n 0 30 Li Parameters Data Transfer Abs T Feak Detection Delete Peak Graph Scale Sample Mumber Save Method Output Options o0090000 9 a tim PUES n 495rm Abs 0 01 d 5 Version 2 1 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 button 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 aro
69. ing the method The saved file may be damaged because it was not closed correctly 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 e To avoid data loss please exit the method first before disconnecting the USB flash drive 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
70. ion 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 Menu Options by pressing Escape L or wait Page 47 70 Qu NanoPhotometer P Class User Manual 5 5 Standard Curve 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 00 for concentration use a reagent blank when required to enter the zero standard The procedure is as follows Parameter Screen Standard Curve Parameters Step 1 Step 2 wavelength Pathlength Step 3 Step 4 Units lt b Ment E Cancel Standard Curve Parameters Step 5 Standard Curve Parameters Step 6 Curve Fit Regression Step 7 Calibration sts a Ment E Cancel Step 9 Version 2 1 Parameter Screen Press 3 to select Functions Press 5 to select Standard Curve Select the Wave
71. ions 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 ox gt 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 pl and pmol ul Enter the Factor using the keypad numbers Default value is 33 range is 0 01 to 9 999 If pmol yl 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 10 Press OK o to 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 displays the results The sample concentration and the ratio of A260 A280 and A260 A230 are c
72. is displayed Repeat for all samples Press Menu Options to display available Options which are described below Press Escape L and confirm with Yes to 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 2 1 Page 37 70 Qu NanoPhotometer P Class User Manual 5 FUNCTIONS Survey of the available Functions Functions e Single wavelength 3 NA haut wavelength UA Absorbance R atio 5 EA Standard Curse 1 Absorbance or T transmission at a Absorbance or T transmission at a single user defined wavelength user Absorbance or T transmission at a single user defined wavelength wavelength Colorimetric assay at a single wavelength based on a simple Factor entered or calculated from a EMEN rans cee soot Spectral plot peeween two user defined wavelengths Range 200 950 nm with user configurable 3 Jmeafindngfuncdo ane 6 sre rT anor at w Seeded veg 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
73. k ug ml ug ul pmol ul mg ml mmol l pmol l g l mg l ug l U I 96 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 Kinetics Parameters 7 98768 even with 1 decimal point selected Press OK to store the chosen parameters OR Cancel LI 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 Press Next to enter the Results screen OR press Cancel E to return to the Parameters screen Version 2 1 Page 46 70 Qu NanoPhotometer P Class User Manual Results Screen a pa a D mn 0 0 D 2 0 4 0 6 0 8 1 0 Ag An dA Slope un Rm 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 1 Parameters DD c mp j Data Transfer Frint D ara Set tO At Cursor Set tn At Cursor Slope Sample Humber Save Method Output Options o00909c Version 2 1 Results Screen Step 12 Insert the reference sample and press Blank key Step 13
74. l Version 2 1 Page 57 70 Qu NanoPhotometer P Class User Manual Direct Data output to PC Connection between NanoPhotometer and PC is possible with a USB or Bluetooth connection Printer Step 1 Select whether Auto print is on or off using the left and right arrows When auto print is set as on the results are automatically printed a after a measurement is taken When setting 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 Step 2 Select how the data are sent Options are Computer USB or Bluetooth E3 Press OK Qo to store the settings and return to the Utilities folder OR Press Cancel to return to the Utilities folder gt OK O Cane without storing the settings Sinter 4 Computer USB b Data output to SD Memory Card Printer Step 1 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 set to 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 Step2 Select printer module SD Memory Card El Press OK to store the settings and return to the Utilities folder OR Press Cancel Q to return to the Utilities folder without storing the settings 4 Computer USB d gt OK Cancel 7
75. 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 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 M to save the method OR Cancel to return to the User Methods folder Version 2 1 Page 55 70 NanoPhotometer P Class User Manual Methods Methods 1 Single wavelength sb Delete Method 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 gt to delete the method OR Cancel Q to return to User Methods folder 7 UTILITIES Survey of the available Utilities Utilities e 1 Set correct time and date Select preferred number format Version 2 1 Page 56 70 Qu NanoPhotometer P Class User Manual 7 141 Date and Time The procedure is as follows Date and Time Step 1 Enter the Day using the keypad numbers or left and right Step2 Enter the Month as above Step 3 Enter
76. lenath Regression NanoPhotometer P 300 Options select using key pad numbers Parameters Frint Graph Edit Sample Pathlength Sample Humber Save Method Printer Settings NanoPhotometer P 330 P360 Menu select using key pad numbers Farameters ata Transfer Edit Sample Farhlenath Sample Humber Save Method Output Options L e o e e Version 2 1 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad Step 22 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 23 Press OK Qo to accept the calibration and go to the Results screen see below OR press Back to return to the Standards screen Results screen Step 24 Insert the reference sample and press Blank key This Will be used for all subsequent samples until changed Step 25 Insert the sample and press Sample Q The concentration of the sample is taken and displayed Step 26 Repeat for all samples Step 27 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
77. length using the keypad numbers or left and right arrows Enter the number of Standard concentration points to be used in the curve 1 9 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 pg ul pmol pl mg ml mmol l mol l g l mg l ug l U I 96 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 L 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 Press Next o to enter the Standards screen OR Press Cancel to cancel selections and return to the Functions folder Page 48 7O Q
78. lid 100 to lid 50 in the software for calculation Concentration too high Dilute sample or change to 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 the software for calculation Concentration too high Dilute sample i Lid Required volume Warning message Instruction O Sample concentration is too low bs too low Abs is too high change to lid Sample concentration is too low or change to lid 5 if zi Abs too low available Abs is too high change to lid Abs too low change to lid J Abs is too high Physical dilution of the sample is necessary or change to lid 100 if available i Abs too low change to lid Abs is too high Physical dilution of the sample is necessary or change to lid 250 if available IT 100 Abs too low change to lid T Abs is too high Physical dilution of the sample is necessary Some of 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 P360 is Lid 10 Lid 50 und Lid 250 Ll un Lm ps E BE 1 aj s prs s s e e LE Lat ru EA 3 E E co Lr Lal Le Lal ru ru ru T T T gt vedere m Lal ru T ss E B Version 2 1 Page 12 70 Qu NanoPhotometer P Class User Manual 4 NANOVOLUME APPLICATIONS AND CUVETTE APPLICATIONS The N
79. low OR press Back to return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Step 18 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 19 Press Replicates to display the replicate entry boxes Use C to clear previously stored results before measuring Step 20 Insert the standard and press Sample Qo 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 input Step 22 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 23 Press Next Qo to accept the calibration and go to the Results screen see below OR press Back Q to return to the Standards screen Page 49 70 Qu NanoPhotometer P Class User Manual Calibration Manual entry Standard Curve Calibration corm uns sem Standard Fathlength Sample Pathlength 00 m 9m Farameters Osta Transfer Edit Sample Farhlenath Sample Humber Save Method Output Options Version 2 1 Calibration Manual entry Step 24 Shows previously entered calibration values and allows values to be entered via the keypad Step 25 The highlighted box can be edited in order to e
80. lows Parameter Screen NanoVolume Applications Frotein l y Parameters Step 1 Step 2 Lid Factor Protein Step 3 PIED Dilution F actor Units Step 17 Background Cuvette Applications Protein UV Parameters Pathlength Protein Dilution Factor Units Background Protein UV Parameters Protein Wavelength Molar Ext coefficient 47 30 Molecular weight B3323 398 ok Protein UW Parameters Step 5 Step 6 Step 7 Protein wavelength Ext coefficient If g cm PER Step 9 2 Cancel Version 2 1 Parameter Screen Press 1 for NanoVolume OR 2 for Cuvette folder Press 2 to select Protein folder Press 1 to select Protein UV mode Using NanoVolume Applications select the Lid Factor as described in the Average Detection Range Sheet or under 3 2 A minimum of 1 5 ul sample volume for lid 10 is recommended 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 Q to cancel the selections and return to the Parameters screen Select whether the Background correction at 320 nm
81. nd go to the Results screen see ES below OR press Back L to cancel selections and return 4 UN NEN to the Standards screen ok Bk Calibration Screen replicates on Step 17 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 18 Press Replicates Qo to display the replicate entry boxes Use C to clear previously stored results before measuring 4 BE Step 19 Insert the standard and press Sample XP to measure the standard and store the result Step 20 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 Step 21 Press OK Qo to accept the calibration and go to the Results screen see below OR press Back to return to the Standards screen Ld D ME OB LO lE L1 E Back Version 2 1 Page 31 70 Qu NanoPhotometer P Class User Manual Calibration Screen manual entry Lowry Calibration 0 200 0 017 A E 0 20 on 0 400 0 056 A Fi 0 600 0 054 A ll 15 0 800 0 132 4 1 000 0 169 A 0 205 A nr Ln DB IH Li Lg LM OK E Back Results screen T50 nm Concentration Absorbance 0 035 4 Curve Fit 0 627 Standard Pathlength Sample Path
82. nd 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 e Calculation of labelled protein concentration C prot Abs 280 Abs 320 A280 factor lid factor dilution factor With dye correction C prot Abs 280 Abs 320 CF aye AbS max aye Abs 320 A280 factor lid factor dilution factor C prot protein concentration mg ml Abs 280 absorbance AU of proteins CF aye dye dependent correction factor at 280 nm to be delivered from dye supplier Abs max dye absorbance at absorption maximum of the dye AU A280 factor molecular weight prot molar extinction coefficient M t cm t prot or 1 extinction coefficient I g cm lid factor virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid e Calculation of fluorescence dye concentration pmol ul C dye ADS max aye Abs 320 lid factor dilution factor aye 10 C dye dye concentration pmol l Abs 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 M cmt Version 2 1 Page 69 70 Qu NanoPhotometer P Class User Manual e Calculation of degree of labelling D P degree of labelling Abs max aye Abs 320 prot
83. ns select the Lid Factor as described in the Average Detection Range Sheet and under 3 2 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 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 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 pl 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 o 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 Q5 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
84. nter 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 26 Press OK Qo to accept the calibration and go to the Results screen see below OR press Back E to return to the Standards screen Results screen Step 27 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 28 Insert the sample and press Sample The concentration of the sample is taken and displayed Step 29 Repeat for all samples Step 30 Press Menu Options to display available options which are described below Step 31 Press Escape L and confirm with Yes MO 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 Printer settings within the method as described in 7 3 Output Options Printer Exit Menu Options by pressing Escape m or wait
85. 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 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 pg ml although this does vary with base composition this can be calculated if the base sequence is known Please refer to 12 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 gt 1 8 and gt 2 0 respectively deviations from this indicate the presence of impurity in the sample but care must be taken in interpretation of results The 260 nm reading is taken near the top of a broad peak in the absorbance spectrum
86. or left and right arrows Step 4 Select the Mode Factor user entered or Standard factor pesca is calculated from a calibration sample using the left Factor and right arrows Step 5 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 I Step 6 if Standard is selected Enter the concentration using o ook S cance keypad numbers Range 0 01 to 9 999 Use the C button to delete the last digit entered Wavelength 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 l pmol ul mg ml mmol l umol l g l mg l ug l U I 96 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 Step 8 To enter the results screen with the selected parameters press Qo OR cancel the selections and return to the Functions folder by pressing Cancel LI Results Screen if using a Factor Results Screen if using a Factor Con
87. ound correction is On Enter the third Wavelength from which the background correction will be obtained Press Next Qo to enter the next screen OR press Cancel to return to the Functions folder Dilution Factor known Enter a Dilution factor by using the keypad numbers within the range 1 00 9 999 Calculate Dilution Factor Press the Menu Options key Enter the Volume of the sample range 0 01 9 999 using the keypad numbers Enter the volume of Diluent range 0 01 9 999 by using the keypad numbers Step 10 Press OK to calculate the dilution factor and return to the Parameters screen OR press Cancel to cancel selections Step 11 Select units of measurement using left and right arrows Options are ug ml ng ul ug pul Step 12 Enter the factor using the keypad numbers Range 0 001 to 9 999 Step 13 Press OK to enter the results screen OR Cancel E to return to the Functions folder Page 53 70 Qu NanoPhotometer P Class User Manual Results Screen Absorbance HR atia 260 nm Sample 0 258 A 1 230 nm R atio 0 166 A Concentration 12 3 war mil Menu select using key pad numbers 1 Parameters Wu Data Transfer F Sample Mumber Save Metrhad E Output Options Version 2 1 Results Screen Step 14 Insert the reference sample Press Blank key This will be
88. 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 96T scale dependent on the Graph Scale option and this is retained for subsequent measurements To zoom out again use the down arrow Step 10 Press Menu Options to display available options which are described next Step 11 Press Escape E and confirm with Yes M to return to the Functions folder Query needs confirmation to avoid unintended escaping the application Page 43 70 Qu NanoPhotometer P Class User Manual Menu select using key pad numbers Farameterz Li ata Transfer Abs ST Fesk Detection Graph Scale Sample Number Save Method Output Options Dogo 000e Peak Detection Shortcut button 4 Peak Detection Auto detect Peaks Peak Detect on Zoom oo ves Min Pk Height Sort Peaks Ey Min Pk width Draw Peaks lt gt OF E Cancel Ww avescan 0 30 0 25 t 0 20 f 045 Lid m dae bun p BOB Leod tite gb be 460 480 500 a je2 e face fae Aes esos e Sample 1 A 45 nm 4bs 0 164 4 Version 2 1 Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options Printer settings 3 Toggle between Absorbance and T mode 4 Displays Peak Detection Parameter Screen See description below
89. placement 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 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 2 1 Page 61 70 Qu 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 flute 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 disinfec
90. rameters ef avelength E45 nm Units a gt Ment Biuret Parameters Regression Calibration 4 Standards Ld Replicates o Ne Standards Screen Biuret Standards Std 3 Std 6 0 600 1 400 gt Newt Version 2 1 Pathlength oS Cancel E Back Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 Step 12 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 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 pmol l g l mg l ug l U I 96 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 Qo to enter the next screen Select the Calibration mode either
91. 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 molybdenum 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 printed out A correlation coefficient of between 0 95 and 1 00 indicates a good straight line Version 2 1 Page 19 70 Qu NanoPhotometer P Class User Manual 4 2 2 Protein UV Method The procedure is as fol
92. riments 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 pe avegepath 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 measurement is taken This can be a maximum of auae 600 seconds 10 minutes VEN Step 5 Duration Enter the time in minutes over which measurements are taken This can be a maximum of 60 minutes Eo ca Step 6 Interval Enter the interval time in seconds between E E canei measurements using the left and right arrows Options are 5 10 15 20 30 or 60 seconds Step 7 Press Next Qo to go to the next parameters screen OR Press Cancel to return to the Functions folder Kinetics Parameters 2 Step8 Select the measurement Mode using the left and right arrows Pathlensth Delta A change in absorbance over the measurement duration or selected period Final A absorbance at the end of the measurement duration or selected time Slope rate of change of absorbance over the ERES measurement duration or selected period Step 9 Units The user can enter a text string up to 8 characters Mode 4 Delta A k long To access a list of pre defined units press the Menu Options key and then use the left right arrows pk 1 Bac
93. rs 5 10 50 100 and 250 Other technical data 2 800 rpm tube size up to 2 0 mi Cuvette storage capacity for eight 10 mm cells Photometric mode Abs T concentration scan ratio multi wavelength kinetics in Abs x factor min SU i40mmx275mmx380mm lt 4 5 kg Operating voltage 90 250 V 50 60 Hz Max 30 VA Output ports Availability of USB flash drive port USB cable SD Memory Card or Bluetooth for easy PC connection and data export on selected models built in printer Input Port USB cable for firmware updates 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 e IMPLEN guarantees that the product supplied has been thoroughly tested to ensure that it meets its published specification The warranty included in the conditions of supply is valid for 12 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 2 1 Page 65 7O Qu NanoPhotometer P Class User Manual 11 SOLVENT COMPATIBILITY Most solvents
94. saved data on the USB flash drive 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 PVC Installation and User Manual Alternatively results may be saved on a SD Memory Card P300 only or sent to the PC via a Bluetooth P300 only 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 e 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 e Note that the light beam is directed from RIGHT to LEFT through the cell chamber therefore please ensure the measurement cell is inserted in the correct alignment e Insert the measurement cell always in the same direction e The cell holder supplied with the instrument accepts the NanoPhotometer P Class Submicroliter Cell and
95. screen 2 Transfer the results via selected Output Options 4 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 LI or wait Page 52 7O Qu 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 Absorbance Ratio Wavelengths wavelength 1 Wavelength 3 wavelength Background Absorbance H atio Parameters Pathlength Factor Dilution Factor 7 000 Units pa ml 1 olume Diluent 0 000 Version 2 1 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 7 to select Absorbance Ratio Enter the first Wavelength by using the keypad numbers or the left and right arrows Enter the second Wavelength as above Select whether a Background correction is applied to both wavelengths 1 and 2 using the left and right arrows If backgr
96. selected wavelength is displayed on the screen Step 10 Use the left and right arrows to move the cursor and display the value at the cursor position 15nm from set wavelength Step 11 Press Menu Options to display available options which are described below Step 12 Press Escape L and confirm with Yes to return to the Functions folder Query needs confirmation to avoid unintended escaping the application folder Page 39 70 Version 2 1 Qu NanoPhotometer P 300 Options select using key pad numbers ui e o e Parameters Print Abst T Frint Graph Sample Mumber Save Method Frinter Settings NanoPhotometer P 330 P 360 Menu select using key pad numbers Farameters Data Transfer Abs ST Print Graph Sample Humber Save Method Output Options Version 2 1 NanoPhotometer P Class User Manual Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle between Absorbance and T mode 4 Print graph greyed out if no data are available T 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
97. standard 10 mm pathlength quartz glass or plastic cells e The optical height of the NanoPhotometer P Class is 15 mm e The minimum volume that can be used is 0 3 ul with the NanoPhotometer P Class Submicroliter Cell e 12mm 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 2 1 Page 6 70 QD 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 IMPLEN LCD Display ON OFF key NANOPHOTOMETER Alphanumeric keys Cellholder Escape Cancel Back Arrow keys Blank Reference Sample Enter selection OK View options On off key Turns the instrument on off Aiowleds Use the four arrow keys to navigate around the display and select the required y 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 uniqu
98. tep 5 Step 6 NanoPhotometer P Class User Manual 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 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 Contrast Erightness Contrast 7 6 About About HanoPhotometer M Serial Humber mi a P1322 W2 2 0 Build 11 Yersion www implen de E OK Version 2 1 Step 1 Step 2 Step 3 Adjust the Brightness using the left and right arrows Adjust the Contrast using the left and right arrows Press OK Utilities folder to store the settings and return to the Displays the instrument serial number and software version Press OK o to close the window and return to the Utilities folder or wait Page 60 7O Qu NanoPhotometer P Class User Manual 8 MAI
99. 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 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 L or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options 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 LI or wait Page 32 70 Qu 7 3 7 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 Pa
100. 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 Sample volume Pathlength Dilution 5 optional 35 5y 2mm i5 10 optional for P300 1 3yl O3 2ul 02mm 150 100 optional 0 3 2ul 1 100 250 optional 0 3 2 yl 1 250 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 lid well with a slightly wet fluff free tissue Use water 70 ethanol or isopropanol Do not use aggressive solvents like strong acids or bases or organic solvents at any time
101. tion N 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 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 X on the product or on the documents accompanying the product indicates that this appliance may not be 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 2 1 Page 62 70 Qu NanoPhotometer P Class User Manual Error messages and Trouble shooting 8 6 Error messages Error text in display Explanation Solution J J If this icon is displayed eXit the application before removing the USB memory stick 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 e USB flash drive was removed before leav
102. turn 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 M 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 to return to the Parameter screen Page 30 7O Qu NanoPhotometer P Class User Manual Calibration Screen replicates off Calibration Screen replicates off Lowry Calibration Step 13 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 ET MEE until changed E Step 14 Insert the standard use C to clear previously stored 0 300 1 400 Te results before measuring Press Sample to measure the standard and store the result mp Ba ma O68 n8 Lo L8 LU lk Step 15 Repeat for all standards A graph will display the results Standards A and the fitted curve as the measurements are made 0 200 0 017 A P4 Use the up and down arrows to select a standard to be 0 400 0 058 A va repeated if a poor reading has been obtained Use C to 0 600 P2 clear the previous reading Din ve Step 16 When all standards are measured press OK Qo to EIT ossa p x accept the calibration a
103. typically used in life science laboratories are compatible with the NanoPhotometer P Class submicroliter cell surface and the lid surfaces The following solvents are compatible for use on the submicroliter cell at room temperature acetone up to 596 acetonitrile benzene butanol carbon tetrachloride chloroform ethanol ether HEPES hexane isopropanol MES methanol methylenchlorid MOPS phenol up to 1 n propanol toluene buffers containing phosphate or chloride salts e g PBS buffer pH 4 10 or citrate or borate low concentrated acids and bases Please note that all kind of high concentrated acids and bases are not recommended It is recommended to wipe off every sample immediately upon completion of a measurement For more specific solvents please contact the Implen support team Support implen de to check the compatibility Version 2 1 Page 66 70 Qu NanoPhotometer P Class User Manual 12 APPENDIX 12 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 nucleic acid concentration ng pl Abs 2
104. u NanoPhotometer P Class User Manual Standard Screen Standard Curve Standards Standard Curve Calibration O10 Li hi eo 30 qn oo Bll o Standard Curve Calibration Replicates H O 0824 mis Ha 0 082 A x ow I ji an 33 n sn En osm Version 2 1 Standards screen Step 10 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 Step 11 Press Next Qo 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 L to return to the Parameter screen Calibration Screen replicates off Step 12 This shows the calibration values and allows standards to be measured Step 13 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 14 Insert the standard use C to clear previously stored results before measuring and press Sample Qo to measure the standard and store the result Step 15 Repeat for all standards A graph will display the results and the fitted curve as the measurements are input Step 16 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 Press OK to accept the calibration and go to the Results screen see be
105. und 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 accepts it and displays the required graph Page 45 70 Qu 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 difference 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 expe
106. 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 L 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 6 P 300 and 7 P330 P 360 Page 15 70 Qu NanoPhotometer P Class User Manual 4 1 3 Analysis of Oligonucleotides The procedure is as follows Parameter Screen NanoVolume Applications Oligo Parameters Lid Factor Units 10 Cuvette Applications Oligo Parameters Fathlenath Units Background Results Screen 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 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 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 Opt
107. y needs confirmation to avoid unintended escaping the application Multi wW avelength m Abs 300 nm 0 015 f 400 nm 0 016 500 nm 0 073 600 nm 0 073 Version 2 1 Page 51 70 Qu NanoPhotometer P 300 Options select using key pad numbers Parameters Print Graph Edit Sample Pathlenath Sample Humber Save Method Printer Settings O90 3506 NanoPhotometer P 330 P360 Menu select using key pad numbers 1 Parameters Data Transfer Edit Sample Farhlenath Save Method Output Options o F Sample Number Version 2 1 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 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 L or wait Menu select using key pad numbers 1 Return to parameters

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