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NanoPhotometer P-Class User Manual Ver. 1.0

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1. 2 800 rpm tube size up to 2 0 ml Cuvette storage capacity for eight 10 mm cells Photometric mode Abs T concentration scan ratio multi wavelength kinetics in Abs x factor min Method storage Up to 81 methods in user methods Built in methods Nucleic acid microarray labelling efficiency protein and cell density Display formats 320 x 240 pixels 140 mm x 275 mm x 380 mm A5 kg Operating voltage 90 250 V 50 60 Hz Max 30 VA Input Output ports SD Memory Card USB or Bluetooth for connection to a PC for direct data download printout and data storage Performance verification Auto diagnostics when switched on Specifications are measured after the instrument has warmed up at a constant ambient temperature and are typical of a production unit As part of our policy of continuous development we reserve the right to alter specifications without notice Warranty e MPLEN 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 1 0 Page 64 68 IMPLEN NanoPhotometer M P Class User Manual
2. 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 Step5 Enter the number of Standard concentration points 1 9 to be used in the curve using the keypad numbers or left and right arrows Step 6 Select Pathlength using the left and right arrows Options are 5 or 10 mm Step 7 Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Menu Options key and then use the left right arrows ug ml ug ul pmol ul mg ml mmol l umol 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 Qo to store the chosen parameters OR Cancel Q Step8 Press Next to enter the next screen Step9 Select the Calibration mode either Standards measure prepared standards Manual keypad data entry or New Standards previous values are blanked new standard can be measured Step 10 if Standards selected Select the number of Replicates using the left and right arrows This determines the number of standards to be measured and averaged at each standard concentration point Can be
3. Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad Step 23 The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9 999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Step 24 Press OK Qo to accept the calibration and go to the Results screen see below OR press Back Q to return to the Standards screen Results screen Step 25 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 26Insert the sample and press Sample W The concentration of the sample is taken and displayed Step 27 Repeat for all samples Step 28 Press Menu Options to display available options which are described below Step 29 Press Escape X 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 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arr
4. NanoPhotometer M P Class User Manual P 300 P 330 P 360 Version 1 0 P 360 300 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 ENG1010 1 2001 Safety requirements for electrical equipment for measurement control and laboratory use General requirements EN561010 2 101 2002 Particular requirements for IVD medical equipment
5. Please contact your local Implen office or an authorized Implen partner for further information Support agreements that help to fulfil the demands of regulatory guidelines concerning GLP GMP calibration certification using filters traceable to international standards during production and quality control certified engineers and calibrated test equipment approved to ISO 9001 standard automatic self diagnostic calibration test during start of the NanoPhotometer P Class result is documented in each data output file possibility to save a PVC file no data manipulation possible hardcopy printout 8 2 Lamp Replacement The xenon lamp should not need replacement until after several years of use In the unlikely event that it does need replacing this should be undertaken by a service engineer from your supplier 8 3 Mixer replacement The mixer should not need replacement until after several years of use In the unlikely event that it does need replacing this should be undertaken by a service engineer from your supplier Caution Do not open the base plate of the vortexer Do not break the seal 8 4 Exchange of the gaiter If the gaiter is damaged please exchange it immediately The gaiter can be ordered separately item number is S 330360 G Caution Switch the instrument off before removing the gaiter Do not look into the gap between the mixer and the housing UV exposure 1 Remove the defect gaiter Version 1
6. 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 1 or 1 extinction coefficient I g cm In the software are the following protein A280 factors pre programmed BSA bovine serum albumin serum albumin mouse and human lysozyme IgG and OD 1 for more information about the factors see 11 3 Protein quantification There is also the possibility to enter custom factors For correct calculation the following settings are needed either the extinction coefficient l g cm or the molar extinction coefficient M4 cm 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
7. Average Detection Range Sheet and under 3 2 Using Cuvette Applications select Pathlength using the left and right arrows Options are 5 mm or 10 mm Select Dilution Factor Units and Factor as described under 4 1 2 Select whether the Dye correction calculation of the dye dependent correction factor is used or not with the left and right arrows The Background correction is always calculated in the Dye methods Select the appropriate Dye Type 10 different AlexaFluors 4 Cy Dyes 6 Oyster Dyes and Texas Red are programmed with their corresponding maximum absorbance wavelength dye dependent correction factor at 260 nm and dye dependent extinction coefficient For further details please refer to 10 2 Nucleic acid fluorescent dye incorporation If using Custom Dye maximum absorbance wavelength of the custom dye dye dependent extinction coefficient and dye dependent correction factor at 260 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 0 000 to 0 999 Page 16 68 AD ye 345 00044 AZEDFAZIO 1 360 NanoPhotometer M P Class User Manual Dye Concentration 0 00 pmol pi Step 9 Step 10 Step 11 Step 12 Step 13 Step 14 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 W This measures at the selected wavelen
8. EN61326 1 2006 Electromagnetic compatibility generic emission standard electrical equipment for measurement control amp laboratory use For further information including unpacking positioning and installation of the products please refer to the user manual Signed Dated October 1 2011 YO SQ Dr Thomas Sahiri Managing Director Implen GmbH Version 1 0 Page 2 68 IMPLEN NanoPhotometer M P Class User Manual TABLE OF CONTENTS 1 ESSENTIAL SAFETY NOT ES c ane onn 4 2 INTRODUCTION E efo e e nee e bn ta aie k n on n e ka en ee web e a n ki ek ote kwe Pe OU enb ape e pen bu pt ann ante 5 2 1 YOUFSp CiITODNOTOM TON ie ii w akl kaki 5 22 Sample handing UD eem api ka lk a e lm a wa e a a e a a a e a l a a a ba 5 2 3 Keypad and display for NanoPhotometer P 300 ccccccsssssseeeseceenseeesecensseeeseoesseeeseoenseeesseoenes 6 2 4 Keypad and display for NanoPhotometer P 330 P 360 cccccssssssseeeseeessneeeeeeeeesseeeseoesseessooeess 7 WSL OPNON S SEE m 8 3 THE NANOPHOTOMETER P CLASS SUBMICROLITER CELL aa aaaeaaaaaasaoooassooonooonnoonoessssonoonnananoannn 9 sl KOCHMICAII IM SWE NON reee rcc 9 32 Sofiware IMSIFUEUONS EE fk e e ki pk emn 10 4 NANOVOLUME AP
9. Parameters Factor 7 000 Pathlength Dilution Factor 1 000 Units p a ml lt I gt OK E Back Absorbance H atio Parameters olume Diluent 0 000 Version 1 0 Parameter Screen Step 1 Press 3 to select Functions Step 2 Press 7 to select Absorbance Ratio Step 3 Enter the first Wavelength by using the keypad numbers or the left and right arrows Step 4 Enter the second Wavelength as above Step 5 Select whether a Background correction is applied to both wavelengths 1 and 2 using the left and right arrows Step 6 If background correction is On Enter the third Wavelength from which the background correction will be obtained Step 7 Press Next Qo to enter the next screen OR press Cancel to return to the Functions folder Step8 Select the Pathlength using the left and right arrows Options are 0 04 0 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications The Pathlength selection has no influence to the result It is just to document the used pathlength Step 9 Dilution Factor known Enter a Dilution factor by using the keypad numbers within the range 1 00 9 999 Step 10 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 11 Press OK to calculate the dilution factor and return to the P
10. The procedure is as follows Step 1 Adjust the Brightness using the left and right arrows Step 2 Adjust the Contrast using the left and right arrows Step3 Press OK Utilities folder Contrast Contrast to store the settings and return to the Version 1 0 Page 58 68 NanoPhotometer P Class User Manual 7 6 About About ManoPhotometerTM Displays the instrument serial number and software version QD SerialNumber 54324 Press OK 2 to close the window and return to the Utilities folder Version 7122220 or walt Build 11 www implen de E OK Version 1 0 Page 59 68 IMPLEN NanoPhotometer M P Class User Manual 8 MAINTENANCE 8 1 Maintenance free Technology The NanoPhotometer M 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
11. use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open Output Options Printer settings possibility to change the Output Printer settings within the method as described in 7 3 Output Options Printer Exit Menu Options by pressing Escape or wait Page 46 68 Version 1 0 IMPLEN NanoPhotometer M 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 Parameter Screen Standard Curve Parameters Step 1 Press 3 to select Functions Step 2 Press 5 to select Standard Curve re alee me en Lu Step 3 Select the Wavelength using the keypad numbers or left and right arrows Step 4 Enter the number of Standard concentration points to be used in the curve 1 9 Ps Step 5 Select the Pathlength using the left and right arrows Options are 0 04 0 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications The Pathlength selection has no influence to t
12. using the left and right arrows Select the Pathlength using the left and right arrows Options are 0 04 0 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications The Pathlength selection has no influence to the result It is just to document the used pathlength To enter the results screen with the selected parameters press OK OR cancel the selections and return to the Functions folder by pressing Cancel Q Results Screen Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 8 Insert sample and press Sample W Step 9 Repeat for all samples Step 10 The result at the selected wavelength is displayed on the screen Step 11 Use the left and right arrows to move the cursor and display the value at the cursor position 15nm from set wavelength Step 12 Press Menu Options to display available options which are described below Step 13 Press Escape and confirm with Yes Qo to return to the Functions folder Query needs confirmation to avoid unintended escaping the application folder Page 38 68 IMPLEN NanoPhotometer P 300 Options select using key pad numbers Parameters Print Abst 38T Print Graph Sample Humber Save Method Printer Settings Doo 3506 NanoPhotometer P 330 P 360 Menu select using key pad numbers Farameters D ata Transfer Abz 3T Print Graph Sample Hum
13. 0 Page 60 68 IMPLEN NanoPhotometer M 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 disinfection Desinfection or cleaning only by wiping no spraying Use no solvents or aggressive chemicals Approved disinfectant solutions Apesin disinfection spray Tana Chemie GmbH Incidin Liquid and Incidin Foam Ecolab Lysoformin Spezial Lysoform Dr Hans Rosemann GmbH Observe all necessary precautions if dealing with hazardous 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 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 carrie
14. 25 Shows previously entered calibration values and allows values to be entered via the keypad Step 26 The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9 999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Step 27 Press OK M to accept the calibration and go to the Results screen see below OR press Back Q to return to the Standards screen Results screen Step 28 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 29 Insert the sample and press Sample W The concentration of the sample is taken and displayed Step 30 Repeat for all samples Step 31 Press Menu Options to display available options which are described below Step 32 Press Escape 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 parameters screen 2 Transfer the results via selected Output Options Printer settings 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open
15. Off 1 2 or 3 Step 11 Press Next Qo to enter the Standards screen OR press Cancel Q to cancel selections and return to the Protein folder Standards Screen Step 12 Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range is 0 001 to 9 999 C button backspaces and clears the last digit entered Step 13 Press Next 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 Q to return to the Parameter screen Page 26 68 IMPLEN NanoPhotometer M P Class User Manual Calibration Screen replicates off Bradford Calibration gib TO MEE 0 400 0 600 L5 0 600 1 0 iad O14 LB ME LO le LH LB e Back Bradford Calibration 0 200 0 058 A Ei 0 400 0 195 A Ed 0 6 0 600 0 330 A 0 800 0462A na P4 1 000 0 5392 A 1 400 0 713 4 fo a y 1 600 D 842 A n ko n a Du BB 18 LU Lg LY LE L i d Calibration Screen replicates on Bradford Calibration Replicates A Replicates 1 z 0 713 3 0 718 A T m E ET 0 718 A 718 A Fa n a n DB GH LU Lu Liy gt Ment ZO Back T E Version 1 0 Calibration Screen replicates off Step 14 This shows the calibration values and allows standards to be measured Insert the reference sampl
16. Output Options Printer settings possibility to change the Output Printer settings within the method as described in 7 3 Output Options Printer Exit Menu Options by pressing Escape or wait Page 49 68 IMPLEN 5 6 Multiple Wavelength NanoPhotometer P Class User Manual This makes up to 5 absorbance measurements on the same sample The procedure is as follows Parameter Screen Multi wavelength Parameters Pathlenath gt OK 300 400 I n DU soo 400 Version 1 0 a3 a OK a Cancel Multi wavelength Parameters A1 ad A2 D Cancel Hulti wavelength 300 nm 400 nm 500 nm 600 nm kulti wavelength ip Ir NJ p Li los Je m n I 1 300 nm 400 nm 500 nm 600 nm Abs 0 0718 0 016 0 0713 0 073 Parameter Screen Step 1 Press 3 to select Functions Step 2 Press 6 to select Multi Wavelength Step 3 Select the number of Wavelengths Step 4 Select the Pathlength using the left and right arrows Options are 0 04 0 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications The Pathlength selection has no influence to the result It is just to document the used pathlength Step 5 Press OK to enter the next screen Step 6 Enter the first Wavelength using either the number keys or the left and right arrows Step 7 Enter the second Wavelength as above and repeat for the number of wavel
17. Version 1 0 Page 3 68 IMPLEN NanoPhotometer M 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 A Caution refer to accompanying documents Background colour yellow symbol and outline black Unpacking Positioning and Installation Check the contents of the package against the delivery note If any shortages are discovered inform your supplier immediately Inspect the instrument for any signs of damage caused in transit If any damage is discovered inform your supplier immediately Ensure your proposed installation site conforms to the environmental conditions for safe operation Indoor use only Temperature range 5 C to 35 C Note that if you use the instrument in a room subjected to extremes of temperature change during the day it may be necessary to recalibrate by switching off and then on again once thermal equilibrium has been established 2 3 hours Maximum relative humidity of 80 up to 31 C decreasing linearly to 50 at 40 C The instrument must be placed on a stable level bench or table that can take its weight lt 4 5 kg so that air can circulate freely around the instrument The equipment should be positioned such that in the event of an emer
18. a e n e a n n a n a n EE a pan oo 58 TO JADOUE pec 59 8 yz nre 60 9 1 Maintenance Tree T cnnolo ty uo ceno ense erae uo cun sana E E EUR In REPE Su NC CEUS KE Eus 60 6 2 Lamp REDIACCIMG IN cte 60 8 9 MIXOF FEDIGCCINGING ske ia ka ki tk a a a km e a ke p a e ak n n a a m n a a e 60 SA EXCHANGE OF NC GANON eun 60 8 5 Cleaning and general care of the instrument ceccseeeeeeenneeeeeeeseeeeeeeeeesseeeseoeaseeeeeooenseeessooenes 61 zn MN ddiu ropomem 62 87 TOUS SAGOU N DR mc m 63 9 AGGESSOR ES eee one about 9 ion 63 10 APPENDIX Goer nee da e ai n pab sp kanon ee ke kota om p te on l en n A ee a a ee a Ann en 65 10 1 NUCISIC acid QUANIINCANION ze ooo soon kile l l mo ka kin s Exe a vet a aj a a aae a ki a a ai Di 65 10 2 Nucleic acid fluorescent dye INCOrPOratiON cccceeecseeeeseeenseeeeeeeesseeeeseeesseeeseoeenseeessooesseesssoeeneeees 65 10 3 Protein QUANNNE ANON ok kika kl l a ka kk a a ok a l ko ik a k e n a kk kim nan 67 10 4 Protein fluorescent dye incorporation kaka anka a aaa aa nannan a nennen enne nenne nnn nnn nnns 67
19. 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 Q OR wait After each measurement the following options are possible in the Menu 1 2 4 T 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 described in 7 3 Output Options Printer Exit Menu by pressing Escape Q OR wait Page 8 68 IMPLEN NanoPhotometer M 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 ul up to 5 ul of undiluted sample Due to a pathlength of O O4 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 sample
20. amount of scatter is affected by the optics of the system distance between the cell holder and instrument exit slit geometry of this slit and the monochromator optics Different spectrophotometer types therefore give different responses for the same turbid sample to compare results they must be normalized using calibration curves A calibration curve can be determined by comparing measured OD 600 to expected OD 600 Expected OD 600 is determined by counting cell number using an alternative technique for example microscope slide method and converting to OD 600 using the rule of thumb that 1 OD 600 5 x 108 cells ml for E Coli Additionally your NanoPhotometer P Class is coming 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 1 0 Page 35 68 IMPLEN NanoPhotometer M P Class User Manual 4 3 2 Analysis of Bacterial Growt
21. are 0 04 0 1 0 2 1 and 2 mm for NanoVolume p a o cence applications and 5 or 10 mm for cuvette applications The Pathlength selection has no influence to the result It is just to document the used pathlength Step 7 To enter the measurements screen with the selected parameters press OK Qo OR cancel the selections and return to the Functions folder by pressing Cancel Q Measurement Screen Measurement Screen Step 8 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed wavescan Step 9 Insert sample and press Sample W Step 10 Repeat for all samples Sample A 450nmi Results Screen Results Screen A graph of the wavescan is displayed along with a table of Absorbance 96T at each peak Up to eight peaks can be E shown Use the left and right arrows to move the cursor oe ls along the graph When it reaches a peak the peak height ea I and width of the peak is displayed at the top of the screen 0 20 To zoom in on the wavelength scale use the up arrow Dance pod A This auto scales on the Absorbance T scale dependent ae i ae ak eae on the Graph Scale option and this is retained for oe j amp ef de subsequent measurements To zoom out again use the down arrow Step 11 Press Menu Options to display available options which are Sample 1 45 nm 4bs 0 164 A described next Step 12 Press Escape and confirm wit
22. dye Abs 320 Formula for Oligonucleotides FOI 9 83 Abs max aye Abs 320 aye 104 Abs 260 Abs 320 With dye correction FOI 9 83 Abs max aye Abs 320 aye 1056 Abs 260 Abs 320 CF aye ADS max aye Abs 320 ADS max dye absorbance at absorption maximum of the dye AU dye dye dependent extinction coefficient M cmt The following dye types and parameters are pre programmed in the NanoPhotometer P Class Dye dependent extinction Dye dependent correction coefficient Eaye factor 260 nm CFpye Alexa Fluor 350 18 400 Alexa Fluor 488 62 000 Alexa Fluor 532 82 300 Dye Type Absorption maximum Dye nm NexFluor660 3660 34107000 Po 660 200000 n all formulas the molar dye dependent extinction coefficient is used Version 1 0 Page 66 68 IMPLEN NanoPhotometer M P Class User Manual 10 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 coeff
23. fluorescence dye concentration pmol Ll C aye ADS max dye Abs 320 lid factor dilution factor aye 10 C dye dye concentration pmol yl ADS max dye absorbance at absorption maximum of the dye AU lid factor virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid E dye dye dependent molar extinction coefficient M cm Version 1 0 Page 6 68 IMPLEN NanoPhotometer M P Class User Manual e Calculation of degree of labelling D P degree of labelling Abs max aye Abs 320 prot 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 ADS max dye absorbance at absorption maximum of the dye AU E prot protein dependent molar extinction coefficient M cm CF aye dye dependent correction factor at 280 nm to be delivered from dye supplier dye dye dependent molar extinction coefficient Mt cmt The following dye types and parameters are pre programmed in the NanoPhotometer P Class Dyes nm coefficient Edye factor 280 nm CFpye 1 2s 32 5 n all formulas the molar dye dependent and the molar protein dependent extinction coefficient is used Version 1 0 Page 68 68
24. 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 Q 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 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 Qo to save the method OR Cancel to return to the User Methods folder Version 1 0 Page 54 68 IMPLEN NanoPhotometer M P Class User Manual Methods Methods 1 e Single wavelength sh De
25. 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 Description NanoPhotometer M 1 Life Science methods such as nucleic acid assays and Sy klancvolume protein assays using the NanoPhotometer M P Class pplicatioris Submicroliter Cell 2 Life Science methods such as nucleic acid assays protein assays and cell density using cuvettes Cuvette pplicationz jus 3 General spectroscopic methods Functions Contains nine folders that can store user adapted Sa methods up to 81 un ser Methods m j Instrument set up date time number format Output Utilities Options and Baseline Compensation set up The instrument is equipped with a standard USB port The NanoPhotometer P Class Software Package is necessary to connect the NanoPhotometer P Class to a PC The software enables the user to print through the PC directly to the printer that is connected to it Data may be stored as Excel spreadsheet report and or table format EMF graphics file a comma delimited csv data file a tab delimited txt data file or in native NanoPhotometer P Class Software format for later access See also NanoPhotometer P Class Installation and User Manual Alternatively results may be saved on a SD Memory Card or sent to the PC via a Bluetooth accessory these can either be
26. measurements are input Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Press OK M to accept the calibration and go to the Results screen see below OR press Back Q to return to the Standards screen Page 33 68 IMPLEN NanoPhotometer M P Class User Manual Calibration Screen manual entry Biuret Calibration 0 2 1 000 0 446 A 1 400 0 542 A hl ye n a a 4 0 6 LH lO lg LM gt e Back C A Results screen E46 nm Concentration Absorbance 0 249 A 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 Settinas DOS F506 NanoPhotometer P 330 P360 Menu select using key pad numbers Farameters Data Transfer Edit Sample Farhlenath Sample Kkunmber Save Method Output Options oo0 O 0G Version 1 0 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad Step 23 The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range is 0 001 to 9 999 Use C to backspace and clear the last digit entered and the up and down arrows to mo
27. 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 a poor reading has been obtained Use C to clear the previous reading Press OK M to accept the calibration and go to the Results screen see below OR press Back Q to return to the Standards screen Page 30 68 IMPLEN NanoPhotometer M P Class User Manual Calibration Screen manual entry Lowry Calibration 0 200 0 017 A i A 0 20 af 0 400 0 056 A a 0 600 0 094 4 te 0 15 y 0 800 0 122 A if 1 000 0 169 A a 11 1 400 0 205 A 3i 0 05 s n a Ln DB LH Ln Ls LM Ju K e Back a Results screen r50 nm ume uem Absorbance Concentration 0 095 A Curve Fit 0 627 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 Settinas
28. samples until changed Step 19 Press Next Qo 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 made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 22 Press OK M to accept the calibration and go to the Results screen see below OR press Back to return to the Standards screen Page 24 68 a Calibration Screen manual entry BCA Calibration 0 057 A m5 0 171 A 0 256 A a 5 0 404 A n e 0 516 A Pi E got 0 627 A A o 0 2 A n a Du Db TE LU le LM E OK E Back Results screen 562 nm Absorbance Concentration O 286 A 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 DOS F506 NanoPhotometer P 330 P360 Menu select using key pad numbers Parameters Osta Transfer Edit Sample Farhlenath Sample Kkunmber Save Method Output Options 68 o c9 Version 1 0 NanoPhotometer P Class User Manual
29. 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 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 12 mm test tubes may be used e g for cell cultures however they are not recommended as higher quality data is produced by using disposable cuvettes for the analysis If used align the indicator line on 12 mm test tubes in the same direction to ensure reproducible positioning of the tube Note that test tubes do not last forever and that the surfa
30. will be used for all subsequent samples until changed Insert the sample and press Sample W The wavelength absorbance and OD6OO value is displayed Repeat for all samples Press Menu Options to display available Options which are described below Press Escape Q 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 1 0 Page 36 68 IMPLEN NanoPhotometer M P Class User Manual 5 FUNCTIONS Survey of the available Functions Functions Single Wavelength yh ma Wavelength Ta Absorbance A atia BO Absorbance or Absorbance or T transmission at a single user defined wavelength transmission at a Absorbance or T transmission at a single user defined wavelength user defined wavelength LE assay at a single wavelength based on a simple Factor entered or calculated from a single standard Spectral plot between two user defined wavelengths Range 200 950 nm with user configurable peak finding function Kinetic colorimetric assay either rate or end value based Colorimetric assay at a single wavelength based on a user programmed curve Absorbance or T transmission at up to 5 user defined wavelen
31. 1 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 ul and ug ul Press OK o to enter the Results screen OR Cancel Q to return to the Protein folder Page 19 68 IMPLEN NanoPhotometer M P Class User Manual Results Screen Results Screen Step 10 Apply insert the reference sample Press Blank Key This will a 2 Protein U be used for all subsequent samples until changed Step 11 Apply insert sample and press Sample W This measures 000 A280 ee at both 260 and 280 nm wavelengths and displays the 000 BO result Protein concentration is calculated corrected by background wavelength value if selected Rest Step 12 If the absorbance value of the sample is not in the linear range a Warning message will pop up and Instruction 13 1 will be displayed in the top left corner of the result screen Please refer to 3 2 Software instructions important EN information on page 11 for further information Um Step 13 Repeat for all samples Step 14 Press Menu Options to display available Options which are described on page 8 Step 15 Press Escape Q 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 pl
32. 10 APPENDIX 10 1 Nucleic acid quantification For determination nucleic acid concentration in solution the absorbance at wavelength 260 nm is used The function describing the concentration to absorbance relation is a modification of the Lambert Beer equation C nuc Abs 260 factor nuc lid factor dilution factor With background correction C nuc Abs 260 Abs 320 factor nuc lid factor dilution factor C nuc nucleic acid concentration ng ul Abs 260 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 10 2 Nucleic acid fluorescent dye incorporation To determine the nucleic acid concentration and the dye concentration after probe labelling a modification of the Lambert Beer equation is used Background correction is always calculated possibility to switch the dye correction on and off 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 dye Abs 320 factor nuc lid factor dilution factor C nuc nucleic acid concentration ng ul Abs 260 absorbance AU of nucleic acids CF aye dye dependent correction factor at 260 nm ADS max dye absorbance at absorption maximum of
33. Acids folder Lid Factor Units Step 3 Press 1 to select dsDNA mode OR 2 to select ssDNA mode OR 3 to select RNA mode Step 4 Using the NanoVolume Applications select the Lid Factor Dilution Factor Factor as described in the Average Detection Range Sheet and under 3 2 Using Cuvette Applications select Pathlength using the left and right arrows Options are Background 5mm or 10 mm Step 5 Enter the Dilution Factor using the keypad numbers Range 1 00 to 9 999 Use the C button to backspace IQ cance e 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 Cuvette Applications 9 999 Enter the volume of the diluent using the keypad dsD NA Parameters numbers Range 0 01 to 9 999 Press OK Qo to calculate the dilution factor and return to the Parameters screen OR press Cancel Q to cancel the selections and return to the Parameters screen Step 6 Background correction at 320 nm is recommended to be n o O Step 7 Select the Units of measurement using the left and right arrows Options ug ml ng ul ug ul Background Step8 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 E Cancel Step 9 Press OK Qo to enter the Results screen OR Cancel amp to return to the Nucleic Acids folder Results Screen Results Screen Step 10 Apply i
34. Applications To determine the degree of labelling the absorbance reading at the wavelength reported for maximum absorbance of the fluorescence dye is used The corresponding extinction coefficient of the dye is used in the Lambert Beer Law to determine the dye concentration c A e d Absorbance values and extinction coefficients are used to calculate the dye per protein ratio For further details please refer to 10 4 Protein fluorescent dye incorporation Colorimetric Bradford Biuret BCA and Lowry protein determination Cuvette Applications The Bradford method depends on quantifying the binding of a dye Coomassie Brilliant Blue to an unknown protein and comparing this binding to that of different known concentrations of a standard protein at 595 nm this is usually BSA bovine serum albumin The Biuret method depends on reaction between cupric ions and peptide bonds in an alkali solution resulting in the formation of a complex absorbing at 546 nm The BCA method also depends on reaction between cupric ions and peptide bonds but in addition combines this reaction with the detection of cuprous ions using bicinchoninic acid BCA giving an absorbance maximum at 562 nm The BCA process is less sensitive to the presence of detergents used to break down cell walls 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 i
35. Calibration Screen replicates off ECA Calibration 2 5 0 400 0 600 L5 0 800 1 0 fia LL DLE CA LU Le LH E Back ECA Calibration DE of 0 200 0 051 A 0 400 0 170 A D 5 d 0 600 0 288 4 EN ai 0 800 0 403 A f l3 P 1 000 0 516 A 1 400 DEZE A the E n a Ln DE DB LO Le LH 3b OK E Back Calibration Screen replicates on BCA Calibration L1 n a n ob OH lo l Liy Version 1 0 Step 14 This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 15 Insert the standard use C to clear previously stored results before measuring Press Sample Qo to measure the standard and store the result Step 16 Repeat for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 17 When all standards are measured press 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 18 This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent
36. Escape from a selection and return to the previous folder Cancel a selection Stop scape Cancel Back 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 Q Enter or confirm a selection Take a measurement Print Prints the results shown on the screen on the built in printer if a built in printer is P 330 and P 360 only connected to the NanoPhotometer Toggle graph on off The graph shows a wavescan plot across the range 220 nm to Graph Data 400 nm for Dye methods 220 nm to 750 nm with cursors denoting 230 260 P 330 and P 360 only 280 and 320 nm Nucleic Acid methods and 260 280 and 320 nm Protein methods Alphanumeric Keys Version 1 0 Page 7 68 IMPLEN 2 5 Menu Options select using key pad numbers Options for P 300 O99 S500 Menu options for P 330 P 360 Parameters Print Graph Frint D ara Only Sample Humber Save Method Printer Settings Parameters Data Transfer Output D ata Only Sample Kumber Save Method Output Options Version 1 0 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
37. PLICATIONS AND CUVETTE APPLICATIONS 1 eeeeeee eee nn enne nennen nnne nnn anna a nnne nnn na sna a nans 12 4 1 Characterization of DNA RNA and Oligonucleotides eeeeee ecce 12 4 1 1 General MfONTAUON ERR 12 4 1 2 Analysis of dsDNA SSDNA and RNA ccccccceeecccceeeeeeeceeeeceeceeeceeceueceesseeecesseaeeeseaeeeessaaeees 14 4 1 3 A Analysis of Oligonucleotides cc cceeccccecceeeeeeeeecaeeeeeeeeeaeeeceeeeseaaeeeeeeesaeaseeeseseeaeeeesessaeeeeeeeeas 15 4 1 4 Dye incorporation for dsDNA ssDNA RNA and Oligonucleotides 16 42 Prolin D TEFIMINAL O Mi kibik ki ou e ii aka Emm 18 4 2 1 general TOR AUOT NEN 18 4 2 2 Protein UV Method cccccccccccceececceececseeeeceeeeeceeeeeseeesseueesseeeessaeeeseaeeeseaeeesaeeesseeessueeessaseesaes 19 4 2 3 Protein UV Dye Method esssssssessseseeeeeeen nnne nnne n nnn nnne nnn nnn nnns nan nnn 21 A 24 BOA ASSAY M danje sonn 23 UST ASSAY kd ki ab kek bek 32 4 3 Bacterial Cell Culture Measurement OD600 ccccessseeeeeenseeseeeseeseeesseeseenseeseonseeseonseeesoees 35 4 3 1 General Information ceccc cc eeecccceeecceceeeeececeeceeceaeueeesseeeceesaeeeeseuuceesseuecess
38. ance values Pressing Cancel ignores the selection pressing OK Qo accepts it and displays the required graph Page 44 68 IMPLEN NanoPhotometer M 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 experiments The procedure to define a new method is as follows Parameter Screen Parameter Screen Kinetics Parameters 1 Step 1 Press 3 to select Functions Step 2 Press 4 to select Kinetics seve lensin Delan Time Step 3 Wavelength Enter all numerical values using the keypad numbers or the left and right arrows l Step 4 Delay time Enter the delay time in seconds bef
39. arameter 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 Using Cuvette Applications select Pathlength using the left and right arrows Options are 5 mm or 10 mm Enter the Dilution Factor using the keypad numbers Range 1 00 to 9 999 Use the C button to backspace and clear the last digit entered OR press Menu Options to enter the dilution factor screen Enter the volume of the sample using the keypad numbers Range 0 01 to 9 999 Enter the volume of the diluent using the keypad numbers Range 0 01 to 9 999 Press OK 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 Select whether the Background correction at 320 nm 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 00
40. arameters screen OR press Cancel Q to cancel selections Step 12 Select units of measurement using left and right arrows Options are ug ml ng ul ug l Step 13 Enter the factor using the keypad numbers Range 0 001 to 9 999 Step 14 Press OK Qo to enter the results screen OR Cancel Q to return to the Functions folder Page 52 68 IMPLEN NanoPhotometer M P Class User Manual Results Screen Absorbance Ratio 60 nm Sample 0 253 A 1 280 nm R atio 1 554 Concentration 12 9 war mil Menu select using key pad numbers d Parameters qu Osta Transfer Sample Mumber Save Method Output Options ooo Version 1 0 Results Screen Step 15 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 16 Insert sample and press Sample W Step 17 Repeat for all samples The absorbance at the selected wavelengths is measured and the ratio between wavelengths 1 and 2 is calculated both corrected by the background wavelength value if this was selected Step 18 Press Menu Options to display available options which are described below Step 19 Press Escape W 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 parameters screen 2 Transfer the results via selected Out
41. ber Save Method Output Options 6090 S606 Version 1 0 NanoPhotometer P Class User Manual Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle between Absorbance and T mode 4 Print graph greyed out if no data are available 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 Q 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 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 Q or wait Page 39 68 IMPLEN NanoPhotometer M P Class User Manual 5 2 Co
42. bration Standards Replicates Standards Screen Biuret Standards Pathlength E Cancel io Cancel 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 Std 3 Std amp 0 600 1 400 Version 1 0 E Back Parameter Screen Press 2 to select Cuvette folder Press 2 to select Protein folder Press 5 to select Biuret mode The default Wavelength setting is 546 nm Enter the number of Standard concentration points 1 9 to be used in the curve using the keypad numbers or left and right arrows Select Pathlength using the left and right arrows Options are 5b mm or 10 mm Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Menu Options key and then use the left right arrows ug ml ug ul pmol ul mg ml mmol l umol 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 gt to enter the next screen Select the Calibration mode either Standards measure prepa
43. ce becomes scratched and blemished through repetitive use if this is the case they should be replaced Version 1 0 Page 5 68 IMPLEN NanoPhotometer M 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 Arrow kevs 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 unique to an application are described in the relevant section of the NanoPhotometer M 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 Back 9 Escape from a selection and return to the previous folder Cancel a selection scape Cancel Back Stop making measurements Set reference to 0 000 A or 100 T on a reference soluti
44. ction pressing OK o accepts them Page 43 68 IMPLEN NanoPhotometer P Class User Manual Add Peak Shortcut button 5 wavescan User Defined Peak A 0 30 E Parameters Date Transfer Abs T Peak Detection Delete Peak Graph Scale Sample Kumber Save Method Output Options a o e O t e Version 1 0 480 S00 A 435nm Abs 0 073 4 Add Peak Shortcut button 5 Adas 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 around the cursor measurement point whilst the other options select the absorbance or wavelength axes respectively With x or y axis limits set to on zooming out will only be permitted to the set limits x y axis limits Setting x or y axis limits to On activates the start and finish points of the desired graph to user defined specific wavelengths and or absorb
45. d display on page 6 P 300 and 7 P330 P 360 Version 1 0 Page 15 68 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 10 2 Nucleic acid fluorescent dye incorporation The procedure is as follows Parameter Screen NanoVolume Applications dsDKHA Dye Parameters Cuvette Applications dsDKHA Dye Parameters Dye Correction Lid Factor Units Dilution Factor Factor Dye Correction Dye Type Pathlength Units Dilution Factor Factor Dye Type Alesa Fluor 350 dsDNxHA Dye Dye Parameters Dye Type Dye Abs Mar Results Screen Version 1 0 Dye Ext Coefficient 10000 Dye Correction 0 000 E Cancel Parameter Screen Press 1 for NanoVolume OR 2 for Cuvette folder Press 1 to select Nucleic Acids folder Press 5 6 7 or 8 to select one of the dye incorporation methods Using the NanoVolume Applications select the Lid Factor as described in the
46. d out in accordance with local environmental regulations for waste disposal Version 1 0 Page 61 68 Qu NanoPhotometer P Class User Manual Error messages and Trouble shooting 8 6 Error messages Error text in display Explanation Calibration failure UV on Reference channel Cell holder obstructed No light on Reference channel Calibration problem UV IR on Reference channel Waiting for software update Keyboard Calibration problem possible lamp failure Submicroliter cell or cuvette in the cell holder by switching on the instrument Instrument was too cold Submicroliter cell or cuvette in the cell holder by switching on the instrument Submicroliter cell or cuvette in the cell holder by switching on the instrument Cell holder has been removed from the instrument and placed back in the wrong position Low light levels The instrument has been turned on and off too fast Insufficient provision of electricity Power supply does not deliver 18V 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 ins
47. e Press Blank key This will be used for all subsequent samples until changed Step 15 Insert the standard use C to clear previously stored results before measuring Press Next Qo to measure the standard and store the result Step 16 Repeat for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 17 When all standards are measured press 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 18 This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 19 Press Replicates Qo 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 made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 22 Press OK M to accept the calibration and go to the Resu
48. e 6 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 1 0 Page 22 68 IMPLEN NanoPhotometer M P Class User Manual 4 2 4 BCA Assay The colorimetric BCA assay is not recommended with the Submicroliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen BCA Parameters wavelength Units cm BCA Parameters Pathlength nen nen IEEE A an an De E Calibration Standards Replicates Version 1 0 Parameter Screen Step 1 Press 2 to select Cuvette folder Step 2 Press 2 to select Protein folder Step 3 Press 2 to select BCA mode Step 4 The default Wavelength setting is 562 nm Step 5 Enter the number of Standard concentration points 1 9 to be used in the curve using the keypad numbers or left and right arrows Step 6 Select Pathlength using the left and right arrows Options are 5 or 10 mm Step 7 Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Menu Options key and then use the left right arrows ug ml ug ul pmol ul mg ml mmol l umol 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 nu
49. ease see 2 3 Keypad and display on page 6 P 300 and 7 P330 P 360 Version 1 0 Page 20 68 IMPLEN NanoPhotometer M P Class User Manual 4 2 3 Protein UV Dye Method The procedure is as follows Parameter Screen NanoVolume Applications Protein Dye Parameters Lid Factor Protein Dilution Factor Units CNN Cuvette Applications Protein Dye Parameters Pathlength Protein Dilution Factor Units Dye Correction cree Protein Dye Dye Parameters Dye Type Dye Abs Max Dye Ext Coefficient SO0000 Dye Correction Version 1 0 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 Lid Factor as described in the Average Detection Range Sheet and under 3 2 A minimum of 1 5 ul sample volume for lid 10 IS recommended Using Cuvette Applications select Pathlength using the left and right arrows Options are 5 mm or 10 mm 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 Param
50. eeeessaeeeessaeeeetsaeees 35 4 3 2 Analysis of Bacterial Growth ccccccscccccsseeeeceseceeseeeeeeeseueceeseaeeeeseaaseeesaeeeesseeeeesseeessaaaeess 36 5 FUNCTION c e e e ea ki 37 5 1 Single Wavelength ADS and Y T kose t sti uua cona pex Enea hieu a ia kf n n ef in n Pa e 38 92 CONC U AON k s s k ak tn non ki ke l ke kk e fi e e ka n ef a n n a e n a a a ei a nn aa 40 BVA OS Cai k wit e asa ca dik vi Je aa l kr e kt n cue a ek kn a E ki a a n e a a ai e ER ai e ka ei kn on den 42 aai aide dey kt ti ot E pk at l n e tn e e kt ar e a ii po el 45 55 Standdrd CUEVO rak tan trase kis sanke aE saki enki dan kk ane a EN onsi fan SANS 47 56 Multiple W AVG IN ise enonse kn iv a ak in ki ka ob aki m re kn kip il pen ek ka ie san ko da ip api sena ke atan Aken 50 57 ADSORB AICS QUO ai io ki ant ki ki mi kk kn an kk aa n a a n e e n op a n n mn a ke ki anl ki a ik ki ie 52 6 USER ME IHODS serpen ne ee ee 54 TNS a E 56 i Datt and TIME sscan a a S 57 T2 Regionale E n e n n A n e ip in n kif kip ae b te 57 TS OUIDUT ODUONS PANN EN ke it ia ra ah n l a e nn eee esse tec e a e n e n e n os pe ia 57 n M i um CS eee ti vie kk eta l wt ik al ft a a kk a a ae a e e e e l e e P dates pk l a e e a n e a e e 58 T9 COMMS occa esas ce cate tcc ii li la n n l e n a e n ei e a n e
51. en use the left right arrows ug ml ug ul pmol ul mg ml mmol l umol l g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed Decimal Points DP to be selected from O to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters OR Cancel Q Press Next gt 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 o to enter the Standards screen OR press Cancel Q to cancel selections and return to the Protein folder Standards Screen Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range 0 001 to 9 999 C button backspaces and clears the last digit entered Press Next Qo to enter the Calibration screen If there are duplicate or non monotonic increasing entries the unit will beep and highlight the incorrect e
52. engths selected up to 5 Step 8 Press OK Qo to enter the results screen OR press Cancel Q to return to the Functions folder Results Screen 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 Step 11 Repeat for all samples A scan plot covering the range of wavelengths selected with cursors at the relevant wavelengths and a table of values is displayed Step 12 Press Menu Options to display available options which are described below Step 13 Press Escape and confirm with Yes to return to the Functions folder Query needs confirmation to avoid unintended escaping the application Page 50 68 IMPLEN NanoPhotometer P 300 Options select using key pad numbers Parameters Frint Graph Edit Sample Pathlength Sample Humber Save Method Printer Settinas ooo F506 NanoPhotometer P 330 P360 Menu select using key pad numbers Farameters Li a Data Transfer A E dit Sample Farhlenath Sample M urnmbser Save Method Output Options os Version 1 0 NanoPhotometer M P Class User Manual Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle graph on off Displays the calibration graph cursors give values for last measured sample 4 Possibility to edit the sample pathlength 7 Samp
53. enter the Standards screen OR Press Cancel Q to cancel selections and return to the Functions folder Version 1 0 Page 47 68 IMPLEN NanoPhotometer M P Class User Manual Standard Screen Standard Curve Standards Standard Curve Calibration 10 0 0 015 4 20 0 0 049 A 30 0 0 082 A ae 0 10 ee 40 0 0 115 4 50 0 0 147 4 Du LI bati 0 1735 Version 1 0 Standards screen Step 11 Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range 0 001 to 9 999 Step 12 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 to return to the Parameter screen Calibration Screen replicates off Step 13 This shows the calibration values and allows standards to be measured Step 14 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 15 Insert the standard use C to clear previously stored results before measuring and press Sample Qo to measure the standard and store the result Step 16 Repeat for all standards A graph will display the results and the fitted curve as the measurements are input Step 17 Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the pre
54. eters screen OR press Cancel Q 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 t 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 Step9 Select the Units of measurement using the left and right arrows Options mg ml ug ml ng ul and ug ul Step 10 Enter the protein dependent extinction coefficient Range is 10 000 to 9 999 999 Step 11 Press OK Qo to store the chosen parameters and to enter the next screen OR Cancel 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
55. factor at 280 nm For further details please refer to 10 4 Protein fluorescent dye incorporation Step 13 If using Custom Dye maximum absorbance wavelength of the custom dye dye dependent extinction coefficient and dye dependent correction factor at 280 nm have to be entered Ranges are Dye Abs Max 300 nm to 950 nm Dye Ext Coefficient 10 000 to 9 999 999 Dye Correction O to 0 999 Page 21 68 Qu Results Screen T 1 3 Protein Dye A280 0 035 A L Protein Concentration ADye 346 0 003 A 464 palm NanoPhotometer P Class User Manual Results Screen 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 Qv 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 0 00 pmol ul Degree of Labelling 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 Escap
56. fer contamination since TRIS EDTA and other buffer salts absorb at this wavelength When measuring RNA samples the 260 230 ratio should be gt 2 0 a ratio lower than this is generally indicative of contamination with guanidinium thiocyanate a reagent commonly used in RNA purification and which absorbs over the 230 260 nm range A wavelength scan of the nucleic acid is particularly useful for RNA samples The instrument can display 260 280 and 260 230 ratios and compensates for dilution and use of cells that do not have 10 mm pathlength dilution factor and cell pathlength can be entered Fluorescent dye incorporation To determine the dye incorporation rate the 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 10 2 Nucleic acid fluorescent dye incorporation Use of Background Correction Background correction at a wavelength totally separate from the nucleic acid and protein peaks at 260 and 280 nm respectively is sometimes used to compensate for the effects of background absorbance The wavelength used is 320 nm and it can allow for the effects of turbidity high absorbance buffer solution and the use of reduced aperture cells If it is
57. gency the mains plug can be easily located and removed This equipment must be connected to the power supply with the power cord supplied It can be used on 90 240 V 50 60 Hz supplies If the instrument has just been unpacked or has been stored in a cold environment it should be allowed to come to thermal equilibrium for 2 3 hours in the laboratory before switching This will prevent calibration failure as a result of internal condensation Switch on the instrument via the keypad after it has been plugged in The instrument will perform a series of self diagnostic checks Please read through this user manual prior to use Please contact your original supplier in the first instance if you experience technical or sample handling difficulties If this equipment is used in a manner not specified or in environmental conditions not appropriate for safe operation the protection provided by the equipment may be impaired and instrument warranty withdrawn Version 1 0 Page 4 68 IMPLEN NanoPhotometer M 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
58. gths Ratio of absorbance values at two user specified wavelengths Menu Options Within each function the user has the possibility to select various options that define the way results are treated If not using a stored method it is advisable to check that these options have been appropriately set for your experiment when coming to the instrument Note that setting the History parameter to On see Preferences later will cause the instrument to store its last settings If the History parameter is turned Off all parameters and selections will return to their default settings when leaving that application Unless it has been saved as a method Version 1 0 Page 3 68 IMPLEN 5 1 Single Wavelength Abs and T NanoPhotometer P Class User Manual This makes simple absorbance A and transmission T measurements on samples measuring the amount of light that has passed through a sample relative to a reference this can be air The procedure is as follows Parameter Screen Single Wavelength Parameters Fathlength Mode Ok wavelength Results Screen Single wavelength 100 Version 1 0 D Cancel Single wavelength Units Abs Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Parameter Screen Press 3 to select Functions Press 1 to select Single Wavelength Set Wavelength by using keypad numbers or left and right arrows Select the Mode Absorbance or Transmission
59. gths and displays the results The sample and dye concentration the FOI and the ratio of A260 A280 and A260 A230 are calculated corrected by the background 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 Q 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 P360 Version 1 0 Page 17 68 IMPLEN NanoPhotometer M P Class User Manual 4 2 4 2 Protein Determination 1 General Information Protein determination at 280 nm NanoVolume Applications and Cuvette Applications Protein can be determined in the near UV at 280 nm due to absorption by tyrosine tryptophan and phenylalanine amino acids Abs 280 varies greatly for different proteins due to their amino acid content and consequently the specific A280 factor for a particular protein must be determined The protein concentration can be calculated the following way C prot Abs 280 A280 factor lid factor
60. h The procedure is as follows Parameter Screen OD 600 Parameters wavelength B00 nm Correction 1 000 c OD 600 Parameters wavelength Factor 86 Correction Multiplier 1 000 4 x 1000 000 k Units de OK e Cancel Results Screen o wemem sammie www m 0 010 A 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 Q to cancel selections and return to the Cuvette Applications folder Results Screen Insert the reference sample and press Blank key This
61. h Yes Qo to return to the Functions folder Query needs confirmation to avoid unintended escaping the application Version 1 0 Page 42 68 IMPLEN NanoPhotometer M P Class User Manual Menu select using key pad numbers Farameters LI ata Transfer Abst BT Peak Detection Graph Scale Sample Number Save Method Output Options Li e e o O e e Peak Detection Shortcut button 4 Peak Detection Auto detect Peaks Peak Detect on Zoom Min Pk Height Sort Peaks By Min Fk width Draw Peaks a OK amp Cancel wavescan 0 30 EM 0 25 f T Liu 0 15 d i 010 14 Lose T FA 0 05 j L CEN Fm mm mak ee 400 420 440 460 460 500 a n favs orsolosse aossors Sample 1 A 450nm 465 0 164 4 Version 1 0 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 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 7 Sample number add a prefix to the sample number and reset the inc
62. he result It is Ment e Cancel Units just to document the used pathlength 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 ul 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 Qo to store the chosen parameters or Cancel Q Standard Curve Parameters Step 7 Select the type of Curve Fit using the left and right arrows Options straight line regression a zero regression this Curve Fit forces the straight line through the origin interpolated or Regression cubic spline Step 8 Select the Calibration mode either Standards measure prepared standards or Manual keypad data entry or Standard Curve Parameters Calibration New Standards previous values are blanked new standard can be measured ugue Step9 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 Ner care Step 10 Press Next P to
63. icient M 4 cm 1 prot oder 1 extinction coefficient Il g cm Abs 260 absorbance AU of nucleic acids lid factor virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid A280 factors pre programmed Serum albumin Serum albumin BSA mouse human IgG Lysozyme 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 cm 1 47 790 43 780 39 310 210 000 37 500 10 4 Protein fluorescent dye incorporation To determine the protein concentration and the dye concentration after labelling a modification of the Lambert Beer equation is used Background correction is always calculated possibility to switch the dye correction on and off 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 dye ADS 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 ADS max dye absorbance at absorption maximum of the dye AU A280 factor molecular weight prot molar extinction coefficient M 1 cm 1 prot oder 1 extinction coefficient Il g cm lid factor virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid e Calculation of
64. ill display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters OR Cancel Q Step 10 Set the Factor by which the result is multiplied to give the amount in the chosen range using the left and right arrows Range of 0 01 to 9 999 Step 11 Select the Pathlength using the left and right arrows Options are 0 04 0 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications The Pathlength selection has no influence to the result It is just to document the used pathlength Step 12 Press Next Qo to enter the Results screen OR press Cancel Q to return to the Parameters screen Version 1 0 Page 45 68 IMPLEN NanoPhotometer M P Class User Manual Results Screen 0 20 Je la I 0 15 I i I 010 el i l l 0 05 i i i L T I a 0 0 D2 o 4 0 6 0g 1 0 Ag A dA Slope ur R 0 001 0 006 0 005 0 099 0 005 0 19557 Sample 1 Time 00 00 10 Abs 0 226 Menu select using key pad numbers 1 Parameters r3 ata Transfer Frint D ata Set tO At Cursor Set tn At Cursor Slope Sample Humber Save Method Output Options o e O t e Results Screen Step 13 Insert the reference sample and press Blank key Step 14 Insert the sample and press Sample to start the run Time min is displayed at the bottom of the screen and absorbance data are plotted on the g
65. ion 1 0 OL e s D4 0 6 n a LO le LM Step 14 Step 15 Step 16 Step 17 Step 18 Step 19 Step 20 Step 21 Step 22 Calibration Screen replicates off This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Insert the standard use C to clear previously stored results before measuring Press Sample Qo 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 L to accept the calibration and go to the Results screen see below OR press Back to cancel selections and return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Press Replicates to display the replicate entry boxes Use C to clear previously stored results before measuring Insert the standard and press Sample Qo 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
66. ion 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 Step 2 Press 1 to select Nucleic Acids folder OR 2 to select Protein folder Step 3 Select the method you want to use by pressing the corresponding number Dilution Factor Factor dsDNA Parameters Step 4 Select the Lid Factor using the left and right arrows Lid amp Samplevolume Pathlength 5 optional 35 5ui 10 optional for P 300 1 3yl Background 63 2p 100 optional 0 3 2yl 1 100 250 optional 0 3 2yl 1 250 Step 5 Select subsequent parameters and specifications as described under 4 Nanovolume Applications and Cuvette Applications After the selections are confirmed the results screen displays in top left corner the chosen Lid and the required sample volume Version 1 0 Page 10 68 IMPLEN NanoPhotometer M P Class User Manual Important Information If the absorbance value of the sample is not in the linear range the following Warning messages will appear and Instruction will be displayed in the top left corner of the result screen Concentration too high Please change to lid 10 and press sample No changes D
67. is 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 Step8 Select the Pathlength using the left and right arrows Options are 0 04 0 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications The Pathlength selection has no influence to the result It is just to document the used pathlength Step9 To enter the results screen with the selected parameters press Qo OR cancel the selections and return to the Functions folder by pressing Cancel Q Results Screen if using a Factor Results Screen if using a Factor Concentration Step 10 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 11 Insert sample and press Sample W Version 1 0 Page 40 68 IMPLEN NanoPhotometer M P Class User Manual Results Screen if using standard mode Concentration Run Standard Concentration Concentration 260 nm z Concentration Absorbance 0 042 A S571 NanoPhotometer P 300 Options select using key pad numbers Parameters Print Graph Run Standard Sample Humber Save Method Printer Settings O98 S309 Nan
68. le 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 Q or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open Output Options settings possibility to change the Output settings within the method as described in 7 3 Output Options Printer Exit Menu by pressing Escape Q or wait Page 51 68 IMPLEN NanoPhotometer M 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 2 Background 2580 nm Co Absorbance Ratio
69. lect how the data are sent Step 2 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Options are Computer USB EZ and depending on the attached printer module SD Memory Card E Bluetooth E3 or None This can also be set using the Menu key in each application or method If None in the Output Options is selected it is only possible to print with the built in printer if it is connected to the instrument For Computer USB SD Memory Card and Bluetooth the auto print option is always on Press OK M to store the settings and return to the Utilities folder OR Press Cancel Q to return to the Utilities folder without storing the settings Select Games function This determines whether the games folder is displayed or not Options are yes or no Define the Screen layout Theme of folders Options are either a grid format or a list Select History whether to use previously entered parameters memory function set On or to return to default settings set Off Select whether to use a 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 M to store the settings and return to the Utilities folder OR press Cancel Q to return to the Utilities folder without storing the settings 7 5 Contrast Ambient temperature can affect the display This function can optimize the display for local conditions
70. lete Method i F3 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 o to delete the method OR Cancel to return to User Methods folder Version 1 0 Page 55 68 IMPLEN NanoPhotometer M P Class User Manual 7 UTILITIES Survey of the available Utilities Utilities o f Jasou Set correct time and date Select preferred number format Printer output options Select screen layout themes history and Baseline Compensation Adjust screen contrast amp brightness Serial number and software version Version 1 0 Page 56 68 IMPLEN 7 1 Date and Time The procedure is as follows Date and Time Day Hour Month Minute Year 2006 gt OK amp Cancel 7 2 Regional Sets Number Format The procedure is as follows Regional Number Format 3955 3 io Cancel 7 3 Output Options Printer Sets up Printer settings P300 The procedure is as follows Auto Print n de OK D Cancel Version 1 0 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 1 Step 2 Step 1 Step 2 Step 3 NanoPhotometer P Class User Manual Enter the Day using the keypad numbers or left and right arrows Enter the Month as above Enter the Year Enter the Hour Enter the Minute Seconds are zeroed when OK is
71. lts screen see below OR press Back Q to return to the Standards screen Page 27 68 IMPLEN NanoPhotometer M P Class User Manual Calibration Screen manual entry Bradford Calibration 0 200 0 058 A A 0 400 0 156 A wi DE y 0 600 0 330 4 te 0 800 0 463 A TA 1 000 x gen 1 400 0 718 A a T S n a DoH E IH Lo lg LM E Back C A gt Results screen Bradford 555 nm Concentration Absorbance 0 337 A 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 Settinas O00 F506 NanoPhotometer P 330 P360 Menu select using key pad numbers Farameters Data Transfer Edit Sample Farhlenath Sample Kkunmber Save Method Output Options oo0 O c9 Version 1 0 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad Step 23 The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range is O 001 to 9 999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Step 24 Press OK Qo to accept the calibration and go to the Results screen see below OR press Back Q to return
72. mber 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 Qo to store the chosen parameters OR Cancel Q Step 8 Press Next to enter the next screen Step 9 Select the Calibration mode either Standards measure prepared standards Manual keypad data entry or New Standards previous values are blanked new standard can be measured Step 10 if Standards selected Select the number of Replicates using the left and right arrows This determines the number of standards to be measured and averaged at each standard concentration point Can be Off 1 2 or 3 Step 11 Press Next Qo to enter the Standards screen OR press Cancel to cancel selections and return to the Protein folder Standards Screen Step 12 Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range 0 001 to 9 999 C button backspaces and clears the last digit entered Step 13 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 Q to return to the Parameter screen Page 23 68 IMPLEN NanoPhotometer M P Class User Manual Calibration Screen replicates off
73. mple is necessary or change to LH 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 lid 250 if available Version 1 0 Page 11 68 IMPLEN NanoPhotometer M P Class User Manual 4 NANOVOLUME APPLICATIONS AND CUVETTE APPLICATIONS The NanoPhotometer P Class offers a complete solution for NanoVolume and standard volume applications With the NanoPhotometer P Class Submicroliter Cell the required sample volume ranges from O 3 ul to a maximal sample volume of 5 ul Standard volume applications can be performed with 10 mm pathlength quartz glass or plastic cuvettes Note Within the Utilities folder the user has the possibility to select various options that define data output please see also 7 3 Output Options Printer The NanoVolume Applications folder and the Cuvette Applications folder contain different sub folders Folder Application Recommended Measurement Cell Nucleic Acids DNA Concentration purity check and dye incorporation for Submicroliter Cell Cuvette DNA samples RNA Concentration purity check and dye incorporation for Submicroliter Cell Cuvette RNA samples Oligo Concentration purity check and dye incorporation for Submicroliter Cell Cuvette Oligo samples Protein Protein UV Protein determination at 280 nm Submicroliter Cell Cu
74. ncentration 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 or left and right arrows Step 4 Select the Mode Factor user entered or Standard factor is calculated from a calibration sample using the left and right arrows EE 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 Step 6 if Standard is selected Enter the concentration using o Ok Came keypad numbers Range 0 01 to 9 999 Use the C button to delete the last digit entered Step 7 Units The user can enter a text string up to 8 characters Concentration Parameters long To access a list of pre defined units press the Menu Options key and then use the left right arrows ug ml ug ul pmol ul mg ml mmol l umol l g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed Th
75. nsert the reference sample Press the Blank Key This will be used for all subsequent samples until changed A230 DO53A Step 11 Apply insert sample and press Sample W This measures A260 0174A at the selected wavelengths and displays the results The sample concentration the ratio of A260 A280 and A320 000A A260 A230 are calculated corrected by the background wavelength value if selected a Step 12 If the absorbance value of the sample is not in the linear range a Warning message will pop up and Instruction will be displayed in the top left corner of the result screen Units Please refer to 3 2 Software instructions important 3 241 information on page 11 for further information Step 13 Repeat for all samples Step 14 Press Menu Options to display available options which are described on page 8 Step 15 Press Escape and confirm with Yes Qo to return to the Nucleic Acids folder AZEDJAZIDO To change parameters print or save methods press the Menu Options button The options menu will be opened For further explanation please see 2 3 Keypad and display on 6 P 300 and 7 P330 P 360 Version 1 0 Page 14 68 IMPLEN NanoPhotometer M P Class User Manual 4 1 3 Analysis of Oligonucleotides The procedure is as follows Parameter Screen NanoVolume Applications Step 1 Oligo Parameters Step 2 Step 3 Lid Factor Units Step 4 Dilution Factor 10 Step 5 1 Cuvette Applicati
76. nto the centre of the measuring window Warning Do not overfill the well Sample volume Pathiength 5 optional 35 5ui 10 optional for P300 1 3 63 2 100 optional 0 3 2yl 1 100 250 optional 0 3 2yl 1 250 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 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 Important Note Residual fluffs must be removed for optimum performance Your cell is ready for the next sample Page 9 68 IMPLEN NanoPhotometer M 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 sect
77. ntry OR press Back Q to return to the Parameter screen Page 29 68 IMPLEN Calibration Screen replicates off Lowry Calibration B5 0 400 0 600 ET La 0 800 fia LL DLE CA LU Le LH E Back Lowry Calibration 0 20 kai 0 200 0 017 A 0 400 0 056 A TA 15 y 0 600 0 095 A ie 0 800 0 132 A ri 010 1 000 0 169 A 1 400 0 205 A A 0 05 pa i p tE Ln ob DA LO Le LH si Ok E Back n a Ln ob OB LO le L4 E Back Version 1 0 Step 14 Step 15 Step 16 Step 17 Step 18 Step 19 Step 20 Step 21 Step 22 NanoPhotometer P Class User Manual 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 Qo 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 L 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
78. o you want to change the lid factor automatic change of lid factor lid 5 to lid 10 in the software for calculation Lid 10 Concentration too low A O Concentration too high Please change to lid 50 and press sample No changes Do you want to change the lid factor automatic change of lid factor lid 10 to lid 50 in the software for calculation Lid 50 Concentration too low Please change to lid 10 apply a minimum of 1pl 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 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 lid 100 to lid 50 in the software for calculation Concentration too high Dilute sample or change to lid 250 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 Required volume Warning message Instruction Abs too low Sample concentration is too low e itu Sample concentration is too low or change to lid 5 if available 3 2 ui Physical dilution of the sample is necessary or change to lid 100 if available 1 2 Physical dilution of the sa
79. oPhotometer P 330 P 360 Menu select using key pad numbers Farameters Osta Transfer Run Standard Sample Humber Save Method Output Options e e o t e Version 1 0 Results Screen if using standard mode Step 12 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 13 Press Sample Qo to display the Run Standard screen Step 14 Run the standard by pressing Run Qo OR Press Cancel Q to return to the measure screen Step 15Insert the sample and press Sample W The concentration of the sample is displayed Results shown as indicate the concentration is out of range Step 16 Repeat for all samples Step 17 Press Menu Options to display available options which are described below Step 18 Press Escape and confirm with Yes Qo to return to the Functions folder Query needs confirmation to avoid unintended escaping the application Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggles on off displaying a graph of wavescan 20 nm from selected wavelength 4 Return to Run Standard screen 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer setting
80. on 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 5O 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 1 0 Page 18 68 IMPLEN NanoPhotometer M P Class User Manual 4 2 2 Protein UV Method The procedure is as follows Parameter Screen NanoVolume Applications Pratein UY Parameters Step 1 Step 2 Lid Factor Protein Step 3 Dilution Factor Units Background Cuvette Applications Protein UV Parameters Pathlength Protein Step 5 Dilution Factor Units Step 6 1 000 Background Step 17 Step 7 Protein LIY Parameters Protein wavelength Molar Ext coefficient 47730 Molecular weight 69323 3295 Protein UWY Parameters Protein wavelength Ext coefficient lf 97cm 0 690 Step 8 Step 9 ae Ok D Cancel Version 1 0 P
81. on at the current Blank Reference wavelength in the mode selected When in scan mode does a reference scan Sample Enter selection OK o Enter or confirm a selection Take a measurement Alphanumeric keys Version 1 0 Page 6 68 IMPLEN NanoPhotometer M P Class User Manual 2 4 Keypad and display for NanoPhotometer P 330 P 360 The back lit liquid crystal display is very easy to navigate around using the alphanumeric entry and navigation arrow keys on the hard wearing spill proof membrane keypad IMPLEN vine E Print with Built in Printer ON Off Key pid Toggle Graph Data menu View Menu NANOPHOTOMETER Alphanumeric Keys Arrow Keys Escape Cancel Back escape Q sample i UE Sample Enter selection OK Blank Reference On Off Key Turns the instrument on off Arrow Kevs Use the four arrow keys to navigate around the display and select the required y setting from the active highlighted option View menu for that application mode Some of these are common to all View Menu applications and described on page 8 Menu unique to an application are described in the relevant section of the NanoPhotometer M 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 Back 9
82. ons Oligo Parameters Step 6 Pathilength Units Step 7 Step 8 Dilution Factor 10 Step 9 10 Step 10 Results Screen Oligo Step 11 Ame oosa Step 12 aso ozea aozo oosa Step 13 Step 14 Step 15 Step 16 Parameter Screen Press 1 for NanoVolume OR 2 for Cuvette folder Press 1 to select Nucleic Acids folder Press 4 to select Oligo mode Using the NanoVolume Applications select the Lid Factor as described in the Average Detection Range Sheet and under 3 2 Using Cuvette Applications select Pathlength using the left and right arrows Options are 5 mm or 10 mm Enter the Dilution Factor using the keypad numbers 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 to cancel the selections and return to the Parameters screen Background correction at 320 nm is recommended to be Switched on Select the Units of measurement using the left and right arrows Options ug ml ng ul ug ul and pmol ul Enter the Factor using the keypad numbers Default value is 33 range is 0 01 to 9 999 If pmol ul is selected there are two options to set the factor 1 A
83. ooo F506 NanoPhotometer P 330 P360 Menu select using key pad numbers Farameters Data Transfer Edit Sample Farhlenath Sample Humber Save Method Output Options oo0 O c9 Version 1 0 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad Step 23 The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range is 0 001 to 9 999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Step 24 Press OK Qo to accept the calibration and go to the Results screen see below OR press Back Q to return to the Standards screen Results screen Step 25 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 26 Insert the sample and press Sample W The concentration of the sample is taken and displayed Step 27 Repeat for all samples Step 28 Press Menu Options to display available Options which are described below press Escape Q and confirm with OK 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 f
84. or last measured sample 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape Q or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 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 or wait Page 31 68 IMPLEN Biuret Assay NanoPhotometer P Class User Manual The colorimetric Biuret assay is not recommended with the Submicroliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen Biuret Parameters Ww auelength Units af Ment Biuret Parameters Biuret Parameters Regression Cali
85. ore the first pete MOU measurement is taken This can be a maximum of 600 seconds 10 minutes Step5 Duration Enter the time in minutes over which Interval measurements are taken This can be a maximum of 60 minutes gt we Step 6 Interval Enter the interval time in seconds between o Non O cane 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 Q to return to the Functions folder Kinetics Parameters 2 Step8 Select the measurement Mode using the left and right arrows Delta A change in absorbance over the measurement duration or selected period Final A absorbance at the end of the measurement duration or selected time Slope rate of change of absorbance over the measurement duration or selected period Factor 5 i Step 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 CMM Bak ug ml ug ul 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 w
86. ows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 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 or wait Page 25 68 IMPLEN NanoPhotometer M P Class User Manual Bradford Assay The colorimetric Bradford assay is not recommended with the Submicroliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen Bradford Parameters Pathlength 555 nm 10 mm Units f Ment e Cancel wW auelength Bradford Parameters Curve Fit Reg i Calibration 4 Standards Ld 6 ne Standards Screen Bradford Standards E Ment E B ack Version 1 0 Parameter Screen Step 1 Press 2 to select Cuvette folder
87. pressed 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 time Set the Number Format decimal point style Options are 44 7 44 77 Or 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 Select whether Auto print is on or off using the left and right arrows When auto print is on the results are automatically printed after a measurement is taken When it is off printing has to be initiated manually This can also be set using the Options key in each application or method The default is OFF Select how the data are sent Options are Computer USB and depending on the attached printer module Built in SD Memory Card E or Bluetooth E3 This can also be set using the Options key in each application or method 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 Page 57 68 IMPLEN Sets up Output options P 330 P360 The procedure is as follows Output Options Output Device amp Cancel 7 4 Preferences Sets user preferences The procedure is as follows Auto Standby Baseline Compensation Preferences History de OR E Cancel NanoPhotometer P Class User Manual Step 1 Se
88. put Options 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 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 or wait Page 34 68 IMPLEN NanoPhotometer M 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 Lag 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
89. put Options Printer settings 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open Output Options Printer settings possibility to change the Output Options Printer settings within the method as described in 7 3 Output Options Printer Exit Menu by pressing Escape Q or wait Page 53 68 IMPLEN NanoPhotometer M P Class User Manual 6 USER METHODS These folders are the storage locations for any user modified Applications Methods that are saved in the Options Menu They are accessible from the home folders page The folder enables the user to quickly select any frequently used Methods Up to 9 Methods may be stored in the folder User Methods ten mee sre oe ki Methods 1 3 gt A Methods 6 PS un PS enu a E i z w li zx M l Mu oo en E a Methods 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 b Folder M ames E Lock Folder Ge Unlock Folder c SD Memon Card Rename Folder Names 1 Press 1 to select Folder Names 2 Select the method to be renamed using the left and right arrows 3 Enter the new name 4 Press OK o to save the new name OR Cancel to return to the User Methods
90. raph as testing proceeds The table below the graph gives absorbance values at Ao start of calculation A finish of calculation dA change in absorbance slope regression parameter R2 of the calculated slope and the result calculated from the selected parameter Step 15 Use the left and right arrows to move the cursor and display the time and absorbance value at measured data points Use the up and down arrows to zoom in or out Step 16 Press Menu Options to display available options which are described below Step 17 Press Escape Q 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 option is greyed out 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method
91. red 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 Q 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 Q to return to the Parameter screen Page 32 68 Qu Calibration Screen replicates off Biuret Calibration 0 400 0 600 0 600 25 T ne 1 0 Biuret Calibration Standards 0 700 o 044 A the NanoPhotometer P Class User Manual wa Ln O86 ON LO l L4 e Back E r X nli c m 14 DE DB LO Le LH 0 400 0 148 A 2 0 600 0 249 A 0 800 0 349 A 0 3 1 000 0 446 4 a 1 400 D 542 A 0 de OK Calibration Screen replicates on Biuret Calibration Replicates 1 0 349 4 the 0 349 A mu 0 345 A UR ha Vers
92. rementing 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 5 w Exit Menu Options by pressing Escape Q 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 the 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 Q ignores the sele
93. rotein impurity The 260 280 ratio gives an indication of purity it is only an indication however and not a definitive assessment Pure DNA and RNA preparations have expected ratios of 1 8 and 2 0 respectively deviations from this indicate the presence of impurity in the sample but care must be taken in interpretation of results The 260 nm reading is taken near the top of a broad peak in the absorbance spectrum for nucleic acids whereas the 280 nm reading is taken on a steep slope i e small changes in wavelength cause large changes in absorbance Consequently small variations in wavelength at 280 nm will have a greater effect on the 260 280 ratio than variations will at 260 nm Thus different instruments of the same and different types may give slightly different ratios due to variations in wavelength accuracy But each instrument will give consistent results within itself Concentration also affects 260 280 readings If a solution is too dilute the readings will be at the instrument s detection limit and results may vary as there is less distinction of the 260 peak and the 280 slope from the Version 1 0 Page 12 68 IMPLEN NanoPhotometer M P Class User Manual background absorbance This is one reason why the Abs 260 value should be greater than O 1 for accurate measurements An elevated absorbance at 230 nm can indicate the presence of impurities as well 230 nm is near the absorbance maximum of peptide bonds and also indicates buf
94. s possibility to change the printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options 4 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 or wait Page 41 68 IMPLEN NanoPhotometer M P Class User Manual 5 3 Wavescan An absorption spectrum can be obtained from your instrument enabling simple identification of peak height and position The procedure is as follows Parameter Screen Parameter Screen wavescan Parameters Step 1 Press 3 to select Functions Step 2 Press 3 to select Wavescan an wa ele oue Step 3 Set Start Wavelength by using keypad numbers or left and right arrows Step 4 Set End Wavelength by using keypad numbers or left and right arrows End Wavelength Step 5 Select the Mode Absorbance or Transmission using the FEFE left and right arrows Step 6 Select the Pathlength using the left and right arrows Options
95. s 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 cr 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 NANOPHOTOMETER P 360 Step 2 Step 3 Step 4 Step 4 Version 1 0 vid 109 IMPLEN 1m Insert the NanoPhotometer P Class Submicroliter Cell into the cell holder with the cell windows facing the light beam We recommend facing the Implen logo to the front The light beam is directed from RIGHT to LEFT as indicated with small arrows Insert the NanoPhotometer P Class Submicroliter Cell always in the same direction Use the integrated vortexer P 330 P 360 only to mix your sample well to achieve an accurate homogeneity of the sample Pipette the appropriate sample volume o
96. 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 108 Press OK Qo to enter the Results screen OR Cancel Q 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 W This measures at the selected wavelengths and displays the results The sample concentration and the ratio of A260 A280 and A260 A230 are calculated corrected by the background wavelength value if selected If the absorbance value of the sample is not in the linear range a Warning message will pop up and Instruction will be displayed in the top left corner of the result screen Please refer to 3 2 Software instructions important information on page 11 for further information Repeat for all samples Press Menu Options to display available Options which are described on page 8 Press Escape Q and confirm with Yes e 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 an
97. 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 aye ADS max aye Abs 320 lid factor dilution factor aye 10 9 C dye dye concentration pmol ul ADS max dye absorbance at absorption maximum of the dye AU lid factor virtual dilution factor 5 10 50 100 and 250 dependent on the used dilution lid 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 1056 Abs 260 Abs 320 With dye correction FOI 6 49 Abs max aye Abs 320 aye 10 Abs 260 Abs 320 CF aye ADS max aye Abs 320 Version 1 0 Page 65 68 IMPLEN NanoPhotometer M P Class User Manual Formula for ssDNA FOI 8 77 AbS max aye Abs 320 aye 105 Abs 260 Abs 320 With dye correction FOI 8 77 Abs max aye Abs 320 dye 10 Abs 260 Abs 320 CF aye ADS max aye Abs 320 Formula for RNA FOI 8 11 Abs max aye Abs 320 dye 10 Abs 260 Abs 320 With dye correction FOI 8 11 Abs max aye Abs 320 E aye 1056 Abs 260 Abs 320 CF dye ADS max
98. ting 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 or wait Page 28 68 IMPLEN NanoPhotometer M P Class User Manual 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 4 Ment E Cancel Lowry Parameters Regression Calibration 4 Standards k Replicates a Standards Screen Lowry Standards gt Ment E Back Version 1 0 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 Step 12 Step 13 Parameter Screen Press 2 to select Cuvette folder Press 2 to select Protein folder Press 4 to select Lowry mode The default Wavelength setting is 750 nm Enter the number of Standard concentration points 1 9 to be used in the curve using the keypad numbers or left and right arrows Select Pathlength using the left and right arrows Options are 5 or 10 mm Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Menu Options key and th
99. to the Standards screen Results screen Step 25 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 26Insert the sample and press Sample W The concentration of the sample is taken and displayed Step 27 Repeat for all samples Step 28 Press Menu Options to display available Options which are described below Step 29 Press Escape and confirm with Yes 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 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Output Options 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incremen
100. trument and start again Remove cuvette or submicroliter cell from the cell holder turn off the instrument and start again Remove the cell holder and place back in the right position If both suggestions doesn t help disconnect the power completely for at least 10 seconds and then reconnect and restart the system again Disconnect the power for quite some time to make sure that the problem is not occurring due to low energy sent to the Xenon flash lamp Disconnect the power completely for at least 10 seconds and then reconnect and restart the system again Check all connections if the cables etc are sticking right Check correct power supply 18 V is attached If possible try another power supply Please contact the Implen Support Team Support implen de Phone 49 89 7263718 20 if none of the mentioned solution helps to solve the problem or if another error message should appear on the NanoPhotometer P Class display Version 1 0 Page 62 68 IMPLEN NanoPhotometer M P Class User Manual 8 7 Trouble shooting Symptom Solution 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 Faul
101. ty or loose power input connection Check voltage output of measurement power supply Thermal paper printer displays random or Paper roll was maybe changed when the instrument is still in one of endless numbers after paper exchange the programs User needs to move to the main menu before exchanging the paper roll Switch the device Off and disconnect the power supply reconnect it after about 5 minutes Firmware update version 2 3 recommended Thermal paper printer prints a plenty of paper Thermal Printer paper was probably put the wrong way check correct normal sound no error message position of the printer paper Thermal printer automatic roller stops Please contact the Implen support team Please contact the Implen Support Team support implen de Phone 49 89 7263718 20 if none of the mentioned solution helps to solve the problem or if another symptom should occur 9 ACCESSORIES photometric accuracy for the NanoPhotometer Version 1 0 Page 63 68 IMPLEN NanoPhotometer M P Class User Manual 11 SPECIFICATION AND WARRANTY Technical Specifications Spectrophotometer Wavelength range 190 1 100 nm Wavelength scan range 200 950 nm System start up time Less than 5 seconas no warm up necessary Measure time for full scan 3 5 seconds range 0 005 A or 1 of the reading whichever is the greater 0 002 Arms at O A 260 nm 0 005 A pk to pk at O A 260 nm NanoVolume application Other technical data
102. used there will be different results from those when unused because Abs 320 is subtracted from Abs 260 and Abs 280 prior to use in equations Concentration Abs 260 Abs 320 Factor Abs ratio Abs 260 Abs 320 Abs 280 Abs 320 Abs ratio Abs 260 Abs 320 Abs 230 Abs 320 If your laboratory has not used background correction before set this option to OFF The use of background correction can remove variability due to handling effects of low volume disposable cells Spectral scan of nucleic acid Pure Nucleic Acid Poly dAdT 0 8 0 7 4 T Wave 260 0 Abs 0 567 T 0 5 o e g 04 Wave 280 0 Abs 0 409 2 5 e a Q os 0 2 0 1 4 0 0 i 210 0 260 0 810 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 1 0 Page 13 68 IMPLEN NanoPhotometer M P Class User Manual 4 1 2 Analysis of dsDNA ssDNA and RNA The procedure is as follows Parameter Screen Parameter Screen NanoVolume Applications dsDNA Parameters Step 1 Press 1 for NanoVolume OR 2 for Cuvette folder Step 2 Press 1 to select Nucleic
103. ve between boxes Step 24 Press OK Qo to accept the calibration and go to the Results screen see below OR press Back Q to return to the Standards screen Results screen Step 25 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 26 Insert the sample and press Sample W The concentration of the sample is taken and displayed Step 27 Repeat for all samples Step 28 Press Menu Options to display available Options which are described below Step 29 Press Escape and confirm with Yes 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 4 Possibility to edit the sample pathlength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Output Options Printer Exit Options by pressing Escape or wait Menu select using key pad numbers 1 Return to parameters screen 2 Transfer the results via selected Out
104. vette Christian Warburg Protein Dye Protein determination at 280 nm and dye incorporation Submicroliter Cell Cuvette BCA Protein determination at 562 nm Cuvette Bradford Protein determination at 595 nm Cuvette Lowry Protein determination at 750 nm Cuvette Biuret Protein determination at 546 nm Cuvette Cell Count ODGOO Cell density at 600 nm Cuvette Nucleic Acids Protein and OD 600 Cell Density Contents of these sub folders are detailed below 4 1 Characterization of DNA RNA and Oligonucleotides 4 1 1 General Information Nucleic Acid Quantification NAQ Nucleic acids can be quantified at 260 nm because it is well established that a solution of dsDNA in a 10 mm pathlength cell with an optical density of 1 0 has a concentration of 50 ug ml ssDNA of 37 ug ml or 40 ug ml in the case of RNA Oligonucleotides have a corresponding factor of 33 ug ml although this does vary with base composition this can be calculated if the base sequence is known Please refer to 10 1 Nucleic acid quantification for further details The instrument uses factors 50 37 40 and 33 as default settings for dsDNA ssDNA RNA and Oligonucleotides respectively and compensation factors for dilution and use of cells which do not have 10 mm pathlength Dilution factor and cell pathlength can be entered Nucleic Acid Purity Checks Nucleic acids extracted from cells are accompanied by protein and extensive purification is required to separate the p
105. vious reading Step 18 Press OK M to accept the calibration and go to the Results screen see below OR press Back Q to return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Step 19 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 20 Press Replicates Qo to display the replicate entry boxes Use C to clear previously stored results before measuring Step 21 Insert the standard and press Sample M to measure the standard and store the result Step 22 Repeat for all replicates and standards A graph will display the results and the fitted curve as the measurements are input Step 23 Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 24 Press Next Qo to accept the calibration and go to the Results screen see below OR press Back Q to return to the Standards screen Page 48 68 IMPLEN NanoPhotometer M P Class User Manual Calibration Manual entry Standard Curye Calibration wavelength i 430 nm ums vom Standard Pathlength Sample Pathlength Menu select using key pad numbers Farameters Data Transfer Edit Sample Farhlenath Sample Kkunmbetr Save Method Output Options oo0 O c9 Version 1 0 Calibration Manual entry Step

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