NanoPhotometer Pearl User Manual Ver. 1.0
Contents
1. Calibration Manual entry Step 24 Shows previously entered calibration values and allows values to be entered via the keypad Step 25 The highlighted box can be edited in order to 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 26 Press OK Qo to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Results screen Step 27 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 28 Insert the sample and press W The concentration of the sample is taken and displayed Step 29 Repeat for all samples Step 30 Press Options to display available Options which are described below Step 31 Press and confirm with M 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 Toggle graph on off Displays 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 fo2. Standard Pathlength Sample Pathlength Options select using key pad numbers Parameters Frint Graph Edit Sample Pathlength Sample Humber Save Method Frinter Settinas ooo F506 Version 1 0 NanoPhotometer M Pearl User Manual 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 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 W The concentration of the sample is taken and displayed Step 27 Repeat for all samples Step 28 Press Options to display available Options which are described below Step 29 Press and confirm with M to return to the Protein folder Query needs confirmation to avoid unintended escaping the application Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle graph on off Displays the calibration graph cu
3. Version 1 0 Results Screen Step 13 Insert the reference sample and press Blank key Step 14 Insert the sample and press to start the run Time min is displayed at the bottom of the screen and absorbance data are plotted on the graph as testing proceeds The table below the graph gives absorbance values at Ao start of calculation An finish of calculation dA change in absorbance slope regression parameter R2 of the calculated slope and the result calculated from the selected parameter Step 15 Use the left and right arrows to move the cursor and display the time and absorbance value at measured data points Use the up and down arrows to zoom in or out Step 16 Press Options to display available Options which are described below Step 17 Press Escape Q and confirm with 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 parameter screen 2 Print data on the results screen via selected method 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 be
4. 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 63 66 IMPLEN NanoPhotometer Pearl User Manual Formula for ssDNA FOI 8 77 AbS max aye Abs 320 dye 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 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 Pearl 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 Alexa Fluor 546 104 000 Alexa Fluor 555 150 000 NexFluor660
5. NanoPhotometer M Pearl User Manual Version 1 0 IMPLEN telephone 49 89 726 3718 O Fax 49 89 726 3718 54 Email info implen de www implen de NanoPhotometer M Pearl User Manual Implen GmbH Schatzbogen 52 D 81829 Germany Declaration of conformity for the NanoPhotometer Pearl This is to certify that the Implen NanoPhotometer Pearl conforms to the requirements of the following Directives 13 23 EEC amp 89 336 EEC Standards to which conformity is declared where relevant are as follows EN 61010 1 2001 Safety requirements for electrical equipment for measurement control and laboratory use EN 61326 2 3 1998 Electromagnetic compatibility generic emission standard Electrical equipment for measurement control and laboratory use EN 61000 4 6 1992 Electromagnetic compatibility generic immunity standard part 1 Residential commercial and light industry For further information including unpacking positioning and installation of the products please refer to the user manual Signed Dated September 1 2010 YG lui Dr Thomas Sahiri Managing Director Implen GmbH Version 1 0 Page 2 66 IMPLEN NanoPhotometer Pearl User Manual TABLE OF CONTENTS 1 ESSEN NALSAFEIY NOTES c c p 4 L1 Unpacking Positioning and Installation iii w ki isk cus ade ki n PR l kt n ARUU e e a RN EE RR DO a a ka SARI M VCU a n kk DO n a a kl RN NAME
6. 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 Qo to display the Run Standard screen Step 14 Run the standard by pressing Qo OR Press Cancel Q to return to the measure screen Step 15 Insert the sample and press 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 Options to display available Options which are described below Step 18 Press and confirm with 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 settings possibility to change the printer settings within the method as described in 7 3 Printer Exit options by pressing Q or wait Page 40 66 IMPLEN NanoPhotometer Pearl User Manual 5 3 Wavescan An absorption spectrum ca
7. Version 1 0 Page 4 66 IMPLEN NanoPhotometer Pearl 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 home page when the instrument is switched on Different folders are numbered and opened by using the associated number key on the keypad After switch on and calibration the default home page is NanoPhotometer offering the choice of HanoPhotometer M 3 Manevalume number nnlicati fasi Life Science methods such as nucleic acid assays and protein assays using the NanoPhotometer Pearl Submicroliter Cell 2 Life Science methods such as nucleic acid assays protein assays and cell density hos Cuvette applications Functions Contains nine folders that can store user adapted methods General spectroscopic methods 7 LI zer Methads ee 5 Instrument set up date time number format and printer and Baseline Compensation set up The instrument is equipped with a standard USB port The NanoPhotometer Pearl Software Package is necessary to connect the NanoPhotometer Pearl 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 sprea
8. by background wavelength value if selected dye concentration and degree of labelling is calculated 1 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 for further information 2 Repeat for all samples 3 Press Options to display available Options which are described on page 8 Press Q and confirm with o to return to the Protein folder 4 w To change parameters print or save methods press the options button The options menu will be opened For further explanation please see 2 3 Keypad and display on page Version 1 0 Page 21 66 IMPLEN NanoPhotometer Pearl 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 Pathlength 562 mm 10 mm 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 righ
9. or 1 extinction coefficient I g cm In the new NanoPhotometer Pearl software are the following protein A280 factors pre programmed BSA bovine serum albumin serum albumin mouse and human lysozyme chicken and IgG mouse 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 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 11 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 Co
10. the diluent using the keypad numbers Range O O1 to 9 999 Press 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 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 mouse or Lysozyme chicken If using Custom Protein there are two possibilities to enter the correct factors Molar extinction coefficient M cm1 Ranges are Wavelength 200 nm to 1 100 nm Molar extinction coefficient M cm t 10 000 to 9 999 999 Molecular weight 0 001 to 9 999 999 Extinction coefficient l g cm Ranges are Wavelength 200 nm to 1 100 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 to enter the Results screen OR Cancel Q to return to the Protein folder Page 18 66 IMPLEN Results Screen EED Protein LI v NanoPhotometer M Pearl User Manual Step 11 Step 12 Step 13 Step 14 Step 15 Step 16 Results Screen Insert the reference sample Press Blank Key This will be used for all subsequent samples until changed Insert sample and press e This measures at both 260 and 280 nm wavelengths an
11. 2 A is being used and ensure that the connector is pushed in fully Instrument switching off after calibration User may be keeping their finger on the ON OFF button too long so that the instrument receives both signals and switches off after the calibration Instrument intermittently switches off during Faulty or loose power input connection Check voltage output of measurement power supply 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 10 ACCESSORIES photometric accuracy for the NanoPhotometer Pearl Version 1 0 Page 61 66 IMPLEN NanoPhotometer Pearl User Manual 11 SPECIFICATION AND WARRANTY Technical Specifications Spectrophotometer Wavelength range 190 1 100 nm Wavelength scan range 200 950 nm System start up time Less than 5 seconds no warm up necessary Measure time for full scan Less than 4 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 lt 4 5 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 af
12. 7 3 Printer Exit options by pressing Q or wait Page 38 66 IMPLEN NanoPhotometer Pearl User Manual 5 2 Concentration This makes simple concentration measurements on samples by measuring the amount of light that has passed through a sample relative to a reference this can be air Concentration is obtained by multiplying the measured absorbance at a Specific wavelength by a factor The factor may be known in advance or may be calculated by the instrument by measuring a standard of known concentration The procedure is as follows Parameter Screen Parameter Screen Concentration Parameters Step 1 Press 3 to select Functions Step 2 Press 2 to select Concentration Step 3 Set Wavelength by using keypad numbers 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 gt ak Step 6 if Standard is selected Enter the concentration using o ok O canei 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 Uoncennauon Poiamerers long To access a list of pre defined units press the Options key and then use the left right arrows ug ml Ug ul pmol ul
13. 8 Save method use the left and right arrows to select a folder to E save Method Store in User Methods 1 9 press the down arrow and enter E Printer Settings name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Printer Exit options by pressing Q or wait Version 1 0 Page 30 66 IMPLEN NanoPhotometer Pearl User Manual 4 2 7 Biuret Assay The colorimetric Biuret assay is not recommended with the Submicroliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen Parameter Screen Biuret Parameters Step 1 Press 2 to select Cuvette folder Step 2 Press 2 to select Protein folder Pathlength Step3 Press 5 to select Biuret mode Step4 The default Wavelength setting is 546 nm Step5 Enter the number of Standard concentration points 1 9 to be used in the curve using the keypad numbers or left PEEESCENES and right arrows TEE Step6 Select Pathlength using the left and right arrows Options are 5 mm or 10 mm E Ment e Cancel Biuret Parameters 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 Options key and then use the left right arrows ug ml Ug ul pmol ul mg dl 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 Point
14. A n 4 2 INTRODUCTION kit t a ie et ta ly tf A a e e a e e e a e n e e ba a e e e 5 2 15 WOUP SPEC ODYO MON NC L TER 5 PS ME elder Atc v t ai e ti vii til iii ki ki esc es eee ec E n an e e e e e e a e e a ee ee e t 5 2 3 TGV AG ANG GS ON AY e ok on poet enn e kenn pt vee cw EE a a a e n SO a a Gi a ea ie ia 6 3 THE NANOPHOTOMETER M PEARL SUBMICROLITER SE ea ei ron tr ti on ron kk ok n kk pk kt RUN E e e n ka n a ke a ak kk ap n n jn l n a 8 S EE I e 20 0 Ei eot mo 8 a2 OPES MSYE UONS iussus isnt at x mea RDUM URINE EFE UMEN ES 9 4 NANOVOLUME APPLICATIONS AND CUVETTE APPLICATIONS 1 e eoe a aa aaa a aaa aaa nenne nnne nn enhn enhn nh nn nanus ana anne annu rans 11 4 1 Characterization of DNA RNA and Oligonucleotides eeeoee eee eee eee eene nennen nnn nnne nnn enhn enhn anna nnn 11 4 1 1 Ce eal WON ci e RE OO oT 11 4 1 2 Analysis ords DNA SSD NA and RNA srcane SRM tit tas tate ARR tt aa n ti atak kk kk TRIN A a n a B C a ap ai 13 4 1 3 Analysis OF Oligonucleotides caine T sa 14 4 1 4 Dye incorporation for dsDNA SSDNA RNA and Oli SONUCIECOTIGES ccccceesseeeeeeeseeeeeeeasseeeeeasseeeeeageeeeesaaeeess 15 4 2 Protein DETCTMINAUON oec 17 4 2 1 Se allt INT GEG a ta kt EEN E l e ei EA E E E ET 17 4 2 2 Protein UV MeMO ki rain in santt tet w aks tel vie ta etd labret a a ak e a a o
15. 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 Wave 260 0 Abs 0 567 0 6 4 0 5 4 0 4 4 Wave 280 0 Abs 0 409 p di Absorbance A 0 3 4 0 2 4 0 1 4 0 0 T 210 0 260 0 310 0 360 0 410 0 Wavelength nm Note e absorbance maximum near 260 nm and absorbance minimum near 230 nm e flat peak near 260 nm and steep slope at 280 nm e very little absorbance at 320 nm Operation of the instrument for Nucleic Acid measurements is described in the following sections DNA and RNA are very similar whilst in Oligo it is possible to calculate the factor from the composite bases by entering the proportions of the 4 bases Version 1 0 Page 12 66 IMPLEN NanoPhotometer Pearl User Manual 4 1 2 Analysis of dsDNA ssDNA and RNA The procedure is as follows Parameter Screen Parameter Screen Step 1 Press 1 for NanoVolume OR 2 for Cuvette folder NanoVolume Applications Step 2 Press 1 to select Nucleic Acids folder dsDNA Parameters Step 3 Press 1 to select dsDNA mode OR 2 to select ssDNA mode OR 3 to select RNA mode Lid Factor Units Step 4 Using the NanoVolume Applications select the Lid Factor as described under 3 2 Using Cuvette Applications select Pathlength using the left and
16. DoH E IH Lo lg LM gt E Back C A Results screen Bradford 555 nm Concentration Absorbance 0 337 A Curve Fit Standard Pathlength Sample Pathlength Regression Options select using key pad numbers Parameters Frint Graph Edit Sample Pathlength Sample Humber Save Method Printer Settinas DOS F506 Version 1 0 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad Step 22 The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9 999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Step 23 Press OK Qo to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Results screen Step 24 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 25 Insert the sample and press W The concentration of the sample is taken and displayed Step 26 Repeat for all samples Step 27 Press Options to display available Options which are described below Step 28 Press and confirm with M to return to the Protein folder Query needs confirmation to avoid unintended escaping the application Options select using key pad numbers 1 Return to pa
17. 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 Parameters screen OR press Cancel to cancel selections Step 12 Select units of measurement using left and right arrows Options are ug ml ng ul ug ul Step 13 Enter the factor using the keypad numbers Range 0 001 to 9 999 Step 14 Press OK Qo to enter the results screen OR Cancel Q to return to the Functions folder Page 51 66 IMPLEN NanoPhotometer Pearl User Manual Results Screen Absorbance Ratio 60 nm Sample 0 253 A 1 280 nm R atio 1 554 Concentration 12 9 war mil Options select using key pad numbers 4 Parameters gt Print E Graph Sample Fumber t E Save Methad Frinter Settings 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 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 Options to display available Options which are described below Step 19 Press W and conf
18. O 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications a gt Ment e Cancel Units 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 Options key and then use the left right arrows ug ml Ug ul pmol ul mg dl 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 o 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 new Standard Curve Parameters Calibration standards using a saved method previous values are blanked new standard can be measured icis Step 9 if standards selected Select the number of standards to be measured and averaged at each standard concentration point Can be OFF 1
19. Range 0 001 to 9 999 C button backspaces and clears the last digit entered Step 12 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 25 66 IMPLEN NanoPhotometer Pearl 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 L6 e Back Bradford Calibration 0 200 0 058 A Ei 0 400 0 195 A Ed 0 6 0 600 0 330 4 0 800 0462A na P4 1 000 0 5392 A 1 400 0 713 8 a 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 13 This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 14 Insert the standard use C to clear previously stored results before measuring Press to measure the standard and store the result Step 15 Repeat for all standards A graph will display the results and the fitt
20. a n l bla m 61 10 PCO Ue See in a ekate pie ta ni e kantite po n e ka ki be ke an n On m ik e kan e e oak be ke ae ian eme 61 MEE Lour aie fwa kek ate et ot pt tr l a at a ki ka ia ee ee L e n pe e e eee E 63 11 1 Nuclei acid GU AN li FO A COI kai ei il Cat tt kk el e e C o 63 11 2 Nucleic acid fluorescent dye incorporation ecole eee e eee aaa nennen nnn nnne nnne ne nnne aene aene aene aene aenea 63 TES ME conii no P Iz ipti erzielen 65 114 Protein Juorescent dye NCONPONA L ON ese cose ens a SE ERES EUR EE a ace KE n a wince ene ec EC FEN ca ERE ank a n ak a a EE Sc Fo E a 65 Version 1 0 Page 3 66 IMPLEN NanoPhotometer Pearl User Manual 1 ESSENTIAL SAFETY NOTES There are a number of warning labels and symbols on your instrument These are there to inform you where potential danger exists or particular caution is required Before commencing installation please take time to familiarise yourself with these symbols and their meaning N Caution refer to accompanying documents Background colour yellow symbol and outline black 1 1 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 environmenta
21. aliae va a ek a e a a a n ak a a a n a a e a e a a e a ee 49 MEE i e LETT 51 6 USER MEN MOD 5 e E E n 53 T CS e E M 55 Ted Date AG MIUERERESOTEUTOTILUTTETTONEENTEUMNEOUUeUe m 56 C EE CION Al EE 56 T 3 PEIDEBRL n ud M jn ki E MED EEUU MU MM MEDIEN RUN RUE 56 re EM i em 57 Ca 6000 RORIS m 57 TO ADO aos estan ec tee 58 8 MAINTENANCE acesterececneecevacetancssives cance EE ates cineca ki ik et e n e e a e e ka ka e kt e a e a a a a e a e e ek kk a a a e ta ea a a et e e kn ta a 59 8 1 Maintenance free Technology 11 aa aaa aaa aa ana aa aaaaeanaaeanooaaenooasononaoenooasenoonsoononasononasonnnnaananaaanonannn 59 8 2 EGRP FE PIO C ANE NT ey ri iii ai kil ki tk kk tk tk nye at a tf ae e ap ae at e ka t ke e nn a e a e p an e e e n e e ae ete ae ae e et a e een e ef er ko e en e e eee 59 5 3 Cleaning and general care Of tie INS UMM si si au ki kwi kk ek ke l in ek k ER Ea ka a a al l ke n a kak a a kk kaa kk e kk kk kt ak kl kl e ek a lk 59 9 ERROR MESSAGES AND TROUBLE SHOOTING teeta tee ieee acters ste es l a aces mee ak l a l ace kt RR ak n a ek e ak ke a pa e a a a je a a EE 60 21 JENON MES SA TE TTE 60 MA TOUIDIS SING O GING e tin vw d tal eli n l e ia a w e ta n e tl e a l ea ct n e e n A n l e a l e n tn n
22. arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Printer Exit options by pressing Q or wait Page 33 66 Qe NanoPhotometer Pearl 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 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
23. 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 Pearl 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 Pearl 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 34 66 IMPLEN NanoPhotometer Pearl User Manual 4 3 2 Analysis of Bacterial Growth The procedure is as follows Parameter Screen OD 600 Parameters wavelength B00 nm Correction 1 000 OD 600 Parameters wavelength E Cancel Factor Correction Multiplier 1 000 4 x 1000 000 k Units celles mil de OK Results Screen 0 010 A E Cancel ce
24. 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 11 66 IMPLEN NanoPhotometer Pearl 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 buffer contamination since TRIS EDTA and other buffer salts absorb at this wavelength When measuring RNA samples the 260 230 ratio should be gt 2 0 a ratio lower than this is generally indicative of contamination with guanidinium thiocyanate a reagent commonly used in RNA purification a
25. maximum absorbance of the fluorescence dye is used For further details please refer to 11 2 Nucleic acid fluorescent dye incorporation The procedure is as follows Parameter Screen NanoVolume Applications Parameter Screen dsD NA Dye Parameters Step 1 Press 1 for NanoVolume OR 2 for Cuvette folder Step 2 Press 1 to select Nucleic Acids folder Lid Factor Jue Step 3 Press 5 6 7 or 8 to select one of the dye incorporation methods Step 4 Using the NanoVolume Applications select the Lid Factor as Dilution Factor Factor described under 3 2 Using Cuvette Applications select 880 Pathlength using the left and right arrows Options are 5 mm or 10 mm Dye Correction Dye Type Step 5 Select Dilution Factor Units and Factor as described under on 4 1 2 Step 6 Select whether the Dye correction calculation of the dye dependent correction factor is used or not with the left and right arrows The Background correction is always calculated in the Dye methods Cuvette Applications Step 7 Select the appropriate Dye Type 10 different AlexaFluors 4 dsDNA Dye Parameters Cy Dyes 6 Oyster Dyes and Texas Red are programmed with their corresponding maximum absorbance wavelength dye Pathlength Units dependent correction factor at 260 nm and dye dependent extinction coefficient For further details please refer to 11 2 Nucleic acid fluorescent dye incorporation Dilution Factor Factor wo Dye Correction Dye Type dsDNA Dye Dye Para
26. 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 S cance Absorbance Ratio Parameters Pathlength Factor 7 000 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 Q to return to the Functions folder Step8 Select the Pathlength using the left and right arrows Options are O 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications Step9 Dilution Factor known Enter a Dilution factor by using the keypad numbers within the range 1 00 9 999 OR Step 10 Calculate Dilution Factor Press the Options key
27. mg dl 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 Qo to store the chosen parameters or Cancel Q Step8 Select the Pathlength using the left and right arrows Options are 0 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications 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 Step 10 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Results Screen if using a Factor Concentration Step 11 Insert sample and press o Version 1 0 Page 39 66 IMPLEN NanoPhotometer Pearl User Manual Results Screen if using standard mode Concentration Run Standard Concentration wavelength 260 nm Concentration Absorbance 0 042 A 150 wal mil S571 Options select using key pad numbers Parameters Print Graph Run Standard Sample Humber Save Method Printer Settings O98 S309
28. 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 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 O O1 to 9 999 Press o to calculate the dilution factor and return to the Parameters screen OR Press Cancel Q to cancel the selections and return to the Parameters screen Background correction at 320 nm is recommended to be switched on Select the Units of measurement using the left and right arrows Options ug ml ng ul ug ul and pmol ul Enter the Factor using the keypad numbers Default value is 33 range is 0 01 to 9 999 If pmol ul is selected there are two options to set the factor 1 A selection table denoting the ratios of the 4 bases according to the oligo sequence Enter the proportions of bases present using the keypad numbers and up and down arrows to move between boxes Default is 10 for each range is O to 9 999 2 Enter the known extinction factor of the oligo used factor range 0 01 to 9 999 for ratio 1 extinction coefficient 10 6 Press OK Qo to enter the Results screen OR Cancel Q to return to the Nucleic Acids folder Results Screen Insert the reference sample Pr
29. 01 to 9 999 Step9 Select the Units of measurement using the left and right arrows Options mg ml ug ml ng ul and ug yl 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 Q to return to the Protein folder Step 12 Select the appropriate Dye Type 4 different AlexaFluors 2 Cy Dyes 2 DyLight Dyes FITC Pacific Blue r PE and Texas Red are programmed with their corresponding maximum absorbance wavelength dye dependent extinction co efficient and dye dependent correction factor at 280 nm 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 For further details please refer to 11 4 Protein fluorescent dye incorporation Ranges are Dye Abs Max 300 nm to 950 nm Dye Ext Coefficient 10 000 to 9 999 999 Dye Correction 0 001 to 0 999 Results Screen Page 20 66 Ga 1 3 Protein Dye AD ye 346 0 003 A d y m palm Degree of Labelling NanoPhotometer Pearl User Manual Results Screen Step 14 Insert the reference sample Press Blank Key This will be used for all subsequent samples until changed Step 15 Insert sample and press W This measures at 260nm 280nm 320nm and the dye specific wavelength and displays the result Protein concentration corrected
30. 2 or 3 Ner care Step 10 Press Next P to enter the Standards screen OR Press Cancel Q to cancel selections and return to the Functions folder Version 1 0 Page 46 66 IMPLEN 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 NanoPhotometer M Pearl User Manual 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 M 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 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 re
31. 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 Pearl 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 66 66
32. 660 107000 Po 660 200000 n all formulas the molar dye dependent extinction coefficient is used Version 1 0 Page 64 66 IMPLEN NanoPhotometer Pearl User Manual 11 3 Protein quantification For determination of protein concentration in solution the absorbance at wavelength 280 nm is used The function describing the concentration to absorbance relation is a modification of the Lambert Beer equation C prot Abs 280 A280 factor lid factor dilution factor With background correction C prot Abs 280 Abs 320 A280 factor lid factor dilution factor C prot protein concentration mg ml Abs 280 absorbance AU of proteins A280 factor Default setting is BSA molecular weight prot molar extinction coefficient M cm 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 Lysozyme Ben mouse human CHE chicken A280 factor 0 78 Molecular Weight g mol 69 323 4 68 692 5 69 365 7 13 850 1 23 237 7 Molar extinction coefficient M cm 1 47 790 43 780 39 310 35 410 29 910 11 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 possib
33. actor as described under 3 2 A minimum of 1 5 ul sample volume 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 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 Qo to calculate the dilution factor and return to the Parameters screen OR Press Cancel to cancel the selections and return to the Parameters screen Step 6 Select whether the Dye correction calculation of the dye dependent correction factor is used or not with the left and right arrows The Background correction is always calculated in the Dye methods Step 7 Select the Protein BSA default Serum Albumin mouse Serum Albumin human IgG mouse or Lysozyme chicken 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 1 100 nm Molar extinction coefficient M 1 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 1 100 nm Extinction coefficient l g cm 0 0
34. ading has been obtained Use C to clear the previous reading Press OK Qo to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Step 18 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 19 Press Qo to display the replicate entry boxes Use C to clear previously stored results before measuring Step 20 Insert the standard and press Qo to measure the standard and store the result Step 21 Repeat for all replicates and standards A graph will display the results and the fitted curve as the measurements are input Step 22 Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 23 Press M to accept the calibration and go to the Results screen see below OR Press Back Q to return to the Standards screen Page 47 66 IMPLEN NanoPhotometer Pearl User Manual Calibration Manual entry Standard Curve Calibration wavelength 430 nm umns vom i Standard Pathlength Sample Pathlength Options select using key pad numbers Parameters Frint Graph Edit Sample Fathlengath Sample urnmber Save Method Printer Settings a a G eo Version 1 0
35. an be printed out A correlation coefficient of between 0 95 and 1 00 indicates a good straight line Version 1 0 Page 17 66 2E NanoPhotometer M Pearl User Manual 4 2 2 Protein UV Method The procedure is as follows Parameter Screen NanoVolume Applications Pratein UY Parameters Step 1 Lid Factor Protein Step 2 A Step 4 Dilution Factor Units Background Cuvette Applications Protein UV Parameters Pathlength Protein Step 6 Dilution Factor Units Step 7 1 000 Background Step 5 Protein LIY Parameters Step 8 Protein wavelength Molar Ext coefficient 47730 Molecular weight 69323 3295 Protein UWY Parameters Protein Wavelength Ext coefficient If 97cm a Step 10 ae Ok D Cancel Version 1 0 Parameter Screen Press 1 for NanoVolume OR 2 for Cuvette folder Press 2 to select Protein folder Press 1 to select Protein UV mode Using NanoVolume Applications select the Lid Factor as described under 3 2 A minimum of 1 5 ul sample volume 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 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
36. ancel a selection Stop making measurements Set reference to 0 000 A or 10096T on a reference solution Blank Reference at the current wavelength in the mode selected When in scan mode does a reference scan Sample Enter selection OK 9 Enter or confirm a selection Take a measurement Alphanumeric keys Version 1 0 Page 6 66 IMPLEN NanoPhotometer Pearl User Manual Options select using key pad numbers Options select using key pad numbers 1 View parameters for the experiments pid meee 2 Print the results Graph 3 Toggle graph on off The graph shows a wavescan plot across the range 220 nm to 400 nm for Dye methods 220 nm to 750 QR Print Data one nm with cursors denoting 230 260 280 and 320 nm 4 Toggle on off the graph in the print out SS T Define the sample number you wish to start from Sample Number 8 Save the parameters as a method to a defined folder name Save Method with a defined method name Printer Settings 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Printer Exit options by pressing Escape Q or wait Experienced operators can use the numeric keys as a shortcut to the option required without needing to enter the Options menu Version 1 0 Page 7 66 IMPLEN NanoPhotometer Pearl User Manual 3 THE NANOPHOTOMETER PEARL SUBMICROLITER CELL With its innovative optical p
37. 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 or to return to default settings Select whether to use a Standby mode after defined periods Options are 1 hour 2 hours at night or off Select Baseline Compensation to improve value stability and to overcome background effects Press OK to store the settings and return to the Utilities folder OR Press Cancel to return to the Utilities folder without storing the settings Ambient temperature can affect the display This function can optimize the display for local conditions The procedure is as follows Contrast Brightness Contrast Version 1 0 Step 1 Step 2 Step 3 Adjust the Brightness using the left and right arrows Adjust the Contrast using the left and right arrows Press OK Utilities folder to store the settings and return to the Page 57 66 NanoPhotometer M Pearl 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 wait Build 11 www implen de E OK Version 1 0 Page 58 66 IMPLEN NanoPhotometer Pearl User Manual 8 MAINTENANCE 8 1 Maintenance free Technology The NanoPhotometer M Pearl technology is maintena
38. athway 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 0 04 mm 0 1 mm 0 2 mm 1 mm and 2 mm the cell is offering an automatic dilution of 1 250 1 100 1 50 1 10 and 1 5 in comparison to a standard cuvette measurement Because the measurements are processed with undiluted samples the reproducibility of the results is samples can be retrieved after the measurement for further processing The NanoPhotometer Pearl Submicroliter Cell can be used for all UV Vis analysis utilizing the wavelength range of 190 to extremely high If desired 1 100 nm The NanoPhotometer Pearl Submicroliter Cell is delivered with two lids for 0 2 mm Lid 50 and 1 mm Lid 10 pathlength which cover most applications Lid 5 2 mm pathlength Lid 100 0 1 mm pathlength and Lid 250 0 04 mm are optional 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 Version 1 0 Step 1 Step 2 Step 3 Step 4 Insert the NanoPhotometer Pearl Submicroliter Cell into the cell holder with the cell windows facing the light beam The light beam is directed from RIGHT to LEFT as indicated with small arrows Insert the NanoPhotometer Pearl Submicroliter Cell always in the same direction Pipette the appropriate sample volume onto the centre of the meas
39. by the background wavelength value if selected a A Step 12 If the absorbance value of the sample is not in the linear la 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 Options to display available Options which are described on page 8 Step 15 Press and confirm with Qo to return to the Nucleic Acids folder AZEDSAZIDO To change parameters print or save methods press the options button The options menu will be opened For further explanation please see 2 3 Keypad and display on page 8 Version 1 0 Page 13 66 IMPLEN NanoPhotometer Pearl 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 Applications Oligo Parameters Step 6 Pathlength Units Step 7 Step 8 Dilution Factor 10 Step 9 10 Step 10 Results Screen Step 11 Step 12 Step 13 Step 14 Step 15 Step 16 Parameter Screen Press 1 for NanoVolume OR 2 for Cuvette folder Press 1 to select Nucleic Acids folder Press 4 to select Oligo mode Using the NanoVolume Applications select the Lid Factor as described
40. ck 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 to display the replicate entry boxes Use C to clear previously stored results before measuring Insert the standard and press 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 to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Page 32 66 IMPLEN NanoPhotometer Pearl 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 Options select using key pad numbers Parameters Frint Graph Edit Sample Pathlength Sample Humber Save Method Printer Settinas DOS F506 Version 1 0 Calibration Screen manual entry Shows previo
41. cted method 3 Toggle between Absorbance and T mode 4 Displays Peak Detection Parameter Screen See description below 5 Manually adds a peak position to the peak table in the results screen at the position set by the cursor If the cursor is returned to this position the legend User Defined Peak is displayed at the top of the scan and this option changes to Delete Peak 6 Displays Graph Scale Parameter Screen See description below 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in Favourites Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Printer Exit options by pressing 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
42. d ab tet e a a kb 18 4 2 3 POS AU DO MI VOL Rm rm 20 4 2 4 BO FPS V a vite ti e r st ie aste tn pt e a n sy ka e n by n Fa r eta n e Ba t f e Aaa t tite Get aa IAN 22 4 2 5 Be AA NON ASE A Yen kita tit ret let eat a a tn la tae a at lett lat it a ced at a tk at a e e ak ao 25 4 2 6 LOWI AS GOY C e l iya pe audi af a n a e e e ek e ki fa kk e kk a at a e a ik n DA H 28 4 2 7 s ESE AV e 31 4 3 Bacterial Cell Culture Measurement ODGOO 11e eoe e eurer eat aseaaaseaseseasesoasosesosoooosoouosoouonoouonoounnonsnnonnnn 34 4 3 1 General TG FAG OD ki vii dey lit cxt Dorn Mass Soc S a ik e A UI QUEE Usu lis Ser RS CE Doch fk ab a a fb a n l n C Un RIDE ni n a 34 4 3 2 Pier S IS Or BSCreHsl Cf ONE pair rwa elt can na av se tk a a ISI AI aU tk ks DIUI UN ISDN UNUS ID e UR E N MM UNO MIS a 35 5 aU pex rec 36 DL Smgl Wavelength ADS and 6Tisssezuaztoiavitis nay Di Ma Rute at ua Ducis ufu Qu can UB dua a a c n ou nb QD e E B Ed 37 s beetle 39 5939 Wav Sta fayit tit ki ik lt kk nAaR ka kf a n la kk a a a tn a ka kk bi a ai 41 ST OM Curie va eti tt ati kl ki ek at e akt n E A a n n t e a e D A 44 o9 undsrd UNE aie rode ie e a ae pe aw n ee kt dik RAM MM 46 55 M ltiple Wavelength 2 a ral vid a a ot ala l al an ki ak va a aaa a a an a
43. d displays the result Protein concentration is calculated corrected by 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 Options to display available Options which are described on page 8 Press and confirm with o to return to the Protein folder To change parameters print or save methods press the options button The options menu will be opened For further explanation please see 2 3 Keypad and display on page 8 Version 1 0 Page 19 66 IMPLEN 4 2 3 Protein UV Dye Method The procedure is as follows Parameter Screen NanoVolume Applications Protein Oye Parameters Lid Factor Protein Dilution Factor Units Cuvette Applications m Cancel Protein Dye Parameters Pathlength Protein Dilution Factor Units Dye Correction e c i Protein Dye Dye Parameters Dye Type Dye Abs FI Dye Ext Coefficient SO0000 Dye Correction Version 1 0 NanoPhotometer Pearl User Manual 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 F
44. d 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 Options key and then use the left right arrows ug ml Ug ul pmol ul mg dl 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 Press Next Qo to enter the next screen Select the Calibration mode either standards measure prepared standards manual keypad data entry or new standards using a saved method 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 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 b
45. dsheet report and or table format EMF graphics file a comma delimited csv data file a tab delimited txt data file or in native NanoPhotometer M Pearl Software format for later access Alternatively results may be saved on a SD Memory Card or sent to the PC via a Bluetooth accessory these can either be supplied pre installed or are available as an optional accessory if the need for the use arises after installation of the product The NanoPhotometer Pearl Software works in a similar way A 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 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 Pearl Submicroliter Cell and standard 10 mm pathlength quartz glass or plastic cells e The optical height of the NanoPhotometer Pearl is 15 mm e he minimum volume that can be used is 0 3 ul with the NanoPhotometer Pearl Submicroliter Cell e 12mm test tubes may be used e g for cell cultures however they are not recommended as higher quality data is produced by using disposable cuvettes for the analysis If used align the ind
46. e 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 36 66 IMPLEN 5 1 Single Wavelength Abs and T NanoPhotometer Pearl 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 Step 1 Step 2 wavelength Step 3 Step 4 Mode Fathlength gt Step 5 Step 6 Ok D Cancel Results Screen Single wavelength Step 7 Sample Step 100 0 a Units 430 440 450 460 410 Single wavelength Units Abs Version 1 0 Parameter Screen Press 3 to select Functions Press 1 to select Single Wavelength Set Wavelength by using keypad numbers or left and right arrows Select the Mode Absorbance or Transmission using the left and right arrows Select the Pathlength using the left and right arrows Options are 0 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications To enter the results screen with the selected parameters press OK OR cancel the selections and return to the Functions folder by p
47. ed curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 16 When all standards are measured press Qo to accept the calibration and go to the Results screen see below OR Press Back to cancel selections and return to the Standards screen Calibration Screen replicates on Step 17 This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 18 Press Qo to display the replicate entry boxes Use C to clear previously stored results before measuring Step 19 Insert the standard and press Qo to measure the standard and store the result Step 20 Repeat for all replicates and standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 21 Press M to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Page 26 66 IMPLEN NanoPhotometer Pearl User Manual Calibration Screen manual entry Bradford Calibration 0 200 0 058 A A 0 400 0 156 A wi 0 6 y 0 600 0 330 A te 0 800 0 463 A e 1 000 x A 1 400 0 718 A a T S n a
48. ee Py and width of the peak is displayed at the top of the screen se a pod A To zoom in on the wavelength scale use the up arrow n m M JG pa Pio This auto scales on the Absorbance T scale dependent oos Pi eem n on the Graph Scale option and this is retained for 400 420 440 480 480 500 subsequent measurements To zoom out again use the Ram LI down arrow Abs o130 D 348 0 086 0 141 DL oq oq Sample 1 A 450nm Abs 0 1644 Step 11 Press Options to display available Options which are described next Step 12 Press and confirm with Qo to return to the Functions folder Query needs confirmation to avoid unintended escaping the application Version 1 0 Page 41 66 IMPLEN NanoPhotometer Pearl User Manual Options select using key pad numbers Parameters Print Abs T Feak Detection Add Peak Graph Scale Sample Purnber Save Method Printer Settings e e o e O e 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 ae OK amp Cancel wavescan 0 30 0 25 a T Li 0 15 HA Lid pe e DANN ERU NS mm i oos i tes Fon mn mak ee 400 420 440 460 460 500 a jazi ese CO favs asosi ae AAA Sample 1 A 450nm 465 0 164 4 Version 1 0 Options select using key pad numbers 1 Return to parameters screen 2 Print result via sele
49. er Query needs confirmation to avoid unintended escaping the application To change parameters print or save methods press the options button The options menu will be opened For further explanation please see 2 3 Keypad and display on page 8 Version 1 0 Page 35 66 IMPLEN NanoPhotometer Pearl User Manual 5 FUNCTIONS Survey of the available Functions Functions Single Wavelength eua Wavelength amp 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 n 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 wavelengths Ratio of absorbance values at two user specified wavelengths 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 th
50. ers Range 4 0 800 i 1 000 A d 0 001 to 9 999 Use C to backspace and clear the last MN Er Ed digit entered and the up and down arrows to move between boxes Step 23 Press OK Qo to accept the calibration and go to the Results screen see below OR Press Back to return to o Back the Standards screen Fi n a Ln DB LH Ln Ls LM Results screen Results screen Step 24 Insert the reference sample and press Blank key This wavelength will be used for all subsequent samples until changed irai Step 25 Insert the sample and press W The concentration of the sample is taken and displayed Step 26 Repeat for all samples Step 27 Press Options to display available Options which are 0 627 described below Press and confirm with Qo to return to the Protein folder Query needs confirmation to avoid unintended escaping the application Absorbance 0 095 A Curve Fit Regression Standard Pathlength Sample Pathlength Options select using key pad numbers Options select using key pad numbers 1 Return to parameters screen 1 Parameters Frin 2 Print result via selected method Grann 3 Toggle graph on off Displays the calibration graph cursors give values for last measured sample Q Edit Sample Pathiensth 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 Sample Number
51. ess Blank Key This will be used for all subsequent samples until changed Insert sample and press 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 Options to display available Options which are described on page 8 Press Q and confirm with o to return to the Nucleic Acids folder To change parameters print or save methods press the options button The options menu will be opened For further explanation please see 2 3 Keypad and display on page 8 Version 1 0 Page 14 66 2E NanoPhotometer M Pearl 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
52. etween 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 entry OR Press Back Q to return to the Parameter screen Page 28 66 IMPLEN Calibration Screen replicates off Lowry Calibration B5 0 400 0 600 ET La 0 800 fia 24 DLE LB LU Le LH E Back Lowry Calibration 0 20 y 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 1563 A 1 400 0 205 A A 0 05 n i p tE 14 BB DA LO Le LH n Ok E Back n a Ln ob OB LO le L4 E Back Version 1 0 Step 13 Step 14 Step 15 Step 16 Step 17 Step 18 Step 19 Step 20 Step 21 NanoPhotometer M Pearl 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 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 obtai
53. icator line on 12 mm test tubes in the same direction to ensure reproducible positioning of the tube Note that test tubes do not last forever and that the surface becomes scratched and blemished through repetitive use if this is the case they should be replaced Version 1 0 Page 5 66 2E NanoPhotometer M Pearl User Manual 2 3 Keypad and display 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 Wo Alphanumeric keys 5 2 a Cellholder Escape Cancel Back Arrow keys A Blank Reference Sample Enter selection OK View options On off key Turns the instrument on off Use the four arrow keys to navigate around the display and Arrow keys select the required setting from the active highlighted option View options for that application mode Some of these are common to all applications and described below Options View Options l a i unique to an application are described in the relevant section 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 l Escape from a selection and return to the previous folder Escape Cancel Back Q C
54. ility 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 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 65 66 IMPLEN NanoPhotometer Pearl 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
55. in 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 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 11 1 Nucleic acid quantification for further details The instrument uses factors 50 37 40 and 33 as default settings for dsDNA ssDNA RNA and Oligonucleotides respectively and compensation factors for dilution and use of cells which do not have 10 mm pathlength Dilution factor and cell pathlength can be entered Nucleic Acid Purity Checks Nucleic acids extracted from cells are accompanied by protein and extensive purification is required to separate the protein impurity The 260 280 ratio gives an indication of purity it is only an indication however and not a definitive assessment Pure DNA and RNA preparations have expected ratios of 1 8 and 2 0 respectively deviations from this indicate the presence of impurity in the sample but
56. irm with 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 Print graph using selected method It is greyed out if no data are available T Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Printer Exit options by pressing Q or wait Page 52 66 IMPLEN NanoPhotometer Pearl 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 E 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 Options select using key pad numbers 1 Folder M ames E Lock Folder Ge Unlock Folder c SD Memon Card Rena
57. l 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 8096 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 4 5 kg so that air can circulate freely around the instrument This equipment must be connected to the power supply with the power cord supplied It can be used on 90 240 V 50 60 Hz supplies If the instrument has just been unpacked or has been stored in a cold environment it should be allowed to come to thermal equilibrium for 2 3 hours in the laboratory before switching This will prevent calibration failure as a result of internal condensation Switch on the instrument via the keypad after it has been plugged in 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
58. lder 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 Printer Exit options by pressing Q or wait Page 48 66 IMPLEN 5 6 Multiple Wavelength NanoPhotometer Pearl 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 n 400 5n a Cancel Multi wavelength Parameters D Cancel 300 nm 400 nm 500 nm 600 nm Multi wavelength Sample q m A M LA Lu NIU DU ann 400 i II 1 L Version 1 0 300 nm 400 nm 500 nm 600 nm 0 015 0 016 0 013 0 013 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 O 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications Step 5 Press OK Qo 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 wavelengths selected up to 5 Step8 Press OK Qo to enter the results screen OR Press Cancel Q to return to the Functions folder Results Scree
59. lls mi 1000 000 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 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 Step 8 Step 9 Step 10 Step 11 Step 12 Step 13 the number of cells per millilitre e g 1 OD 600 5 x 108 cells ml Press OK o to enter the Results screen OR Press Cancel to cancel selections and return to the Cuvette Applications folder Results Screen Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Insert the sample and press W The wavelength absorbance and OD600 value is displayed Repeat for all samples Press Options to display available Options which are described below Press and confirm with Qo to return to the Cuvette Applications fold
60. me 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 o to save the new name OR to return to the User Methods folder Lock Method 1 Press 2 to select Lock Folder 2 Select the method to be locked using the left and right arrows 3 Select a pass code using the keypad numbers or left and right arrows 4 Press o to lock the method OR 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 o to unlock the method OR 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 Pearl 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 Qo to save the me
61. meters Step 8 If using Custom Dye maximum absorbance wavelength of the custom dye dye dependent extinction coefficient and Dye Type Dye Abs Max dye dependent correction factor at 260 nm have to be entered Ranges are Dye Ext Coefficient Dye Abs Max 300 nm to 950 nm Dye Ext Coefficient 10 000 to 9 999 999 Dye Correction 0 000 to 0 999 Dye Correction 0 000 om Results Screen Version 1 0 Page 15 66 Step 9 Step 10 Aee oaza Step 11 Dye Concentration AZEDFAZIO 1 360 0 00 pmol pi Step 12 Step 13 Step 14 NanoPhotometer M Pearl User Manual Results Screen Insert the reference sample Press Blank Key This will be used for all subsequent samples until changed Insert sample and press This measures at the selected wavelengths 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 Options to display available Options which are described on page 8 Press Q and confirm with o to return to the Nucleic Acids folder To change parameters print or save
62. methods press the options button The options menu will be opened For further explanation please see 2 3 Keypad and display on page 8 Version 1 0 Page 16 66 IMPLEN NanoPhotometer Pearl 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 see also application sheet Protein Formulas The protein concentration can be calculated the following way C prot Abs 280 A280 factor lid factor dilution factor With background correction C prot Abs 280 Abs 320 A280 factor lid factor dilution factor This equation can be applied to other proteins if the corresponding factors are known please note that the factor used by the NanoPhotometer Pearl 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 cm
63. mp y axis expands the display around the cursor measurement point whilst the other options select the absorbance or wavelength axes respectively With x or y axis limits set to on zooming out will only be permitted to the set limits X y axis limits Setting x or y axis limits to On activates the start and finish points of the desired graph to user defined specific wavelengths and or absorbance values Pressing Cancel ignores the selection pressing Qo accepts it and displays the required graph Page 43 66 IMPLEN NanoPhotometer Pearl 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 releva
64. n Step9 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 10 Insert sample and press 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 Options to display available Options which are described below Step 13 Press and confirm with Qo to return to the Functions folder Query needs confirmation to avoid unintended escaping the application Page 49 66 IMPLEN NanoPhotometer Pearl User Manual Options select using key pad numbers Options select using key pad numbers Parsmeters 1 Return to parameters screen Frin 2 Print result via selected method 4 Print graph using selected method It is greyed out if no data are available Q Print Graph 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 E Sample Number store in User Methods 1 9 press the down arrow and enter Save Methad name Printer Settings 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Printer Exit options by pressing Q or wait Version 1 0 Page 50 66 IMPLEN 5 7 Absorbance Ratio NanoPhotometer Pearl User Manual This makes simple absorbance ratio
65. n 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 PEPE left and right arrows Step 6 Select the Pathlength using the left and right arrows Options are O 1 0 2 1 and 2 mm for NanoVolume ok ak E cancel applications and 5 or 10 mm for cuvette applications mE v 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 WERE Measurement Screen Step 8 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed 2 0 Step 9 Insert sample and press 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 A Absorbance T at each peak Up to eight peaks can be ee hi shown Use the left and right arrows to move the cursor ki f along the graph When it reaches a peak the peak height ae e
66. nce 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 Pearl secondary standards as an optional accessory These NanoPhotometer Pearl Didymiumglasfilters are suitable for the control and documentation of the wavelength accuracy and the photometric accuracy of your system Please contact your local Implen office or an authorized Implen partner for further information 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 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 M Pearl design edition glossy anthracite All painted surfaces of the NanoPhotometer Pearl can be cleaned with a soft damp cloth and approved cleaning or disinfectant solutions Caution Product damage by wrong cleaning or disinfection N Desinfection o
67. nd 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 11 2 Nucleic acid fluorescent dye incorporation Use of Background Correction Background correction at a wavelength totally separate from the nucleic acid and protein peaks at 260 and 280 nm respectively is sometimes used to compensate for the effects of background absorbance The wavelength used is 320 nm and it can allow for the effects of turbidity high absorbance buffer solution and the use of reduced aperture cells If it is used there will be different results from those when unused because Abs 320 is subtracted from Abs 260 and Abs 280 prior to use in equations Concentration Abs 260 Abs 320 Factor Abs ratio Abs 260 Abs 320 Abs 280 Abs 320 Abs ratio Abs 260 Abs 320 Abs 230
68. ned Use C to clear the previous reading When all standards are measured press Qo to accept the calibration and go to the Results screen see below OR Press Back Q to cancel selections and return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Press Qo to display the replicate entry boxes Use C to clear previously stored results before measuring Insert the standard and press 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 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 Qo to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Page 29 66 a NanoPhotometer M Pearl User Manual Calibration Screen manual entry Calibration Screen manual entry Lowry Calibration Shows previously entered calibration values and allows 3 values to be entered via the keypad sinus ue Step 22 The highlighted box can be edited in order to enter an 0 400 0 056 A ET oe absorbance value corresponding to a given ins ra concentration value using the keypad numb
69. nge 0 001 to 9 999 C button backspaces and clears the last digit entered Step 13 Press Next to enter the Calibration screen If there are duplicate or non monotonic increasing entries the unit will beep and highlight the incorrect entry OR Press Back Q to return to the Parameter screen Page 22 66 IMPLEN NanoPhotometer Pearl User Manual Calibration Screen replicates off Calibration Screen replicates off ECA Calibration 2 5 0 400 0 600 L5 0 800 1 0 fia 24 DLE LB LU Le LH E Back ECA Calibration DE of 0 200 0 051 A A 0 400 0 170 A D 5 i 0 600 D 288A my md 0 800 0 403 A l3 y 1 000 0 516 A 1 400 DEZE A the E n a 14 DE DB LO Le LH si OK E Back Calibration Screen replicates on BCA Calibration LE T MM 1 0 626 A A 2 06264 ms ye i 1 400 0 626 A Fa a 3 d n p Fi D1 Ta 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 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 standa
70. nt 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 before the first oo 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 to return to the Functions folder Kinetics Parameters 2 Step 8 Select the measurement Mode using the left and right arrows Delta A change in absorbance over the measurement duration or selected period Final A absorbance at the end of the measurement duration or selected time Slope rate of change of absorbance over the measurement duration or selected period Factor 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 e Options key and then use the left right arr
71. o 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 77 44 77 Or Press OK Qo to store the settings and return to the Utilities folder OR Press Cancel to return to the Utilities folder without storing the settings 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 56 66 IMPLEN 7 4 Preferences Sets user preferences The procedure is as follows Preferences Games Auto Standby or Theme Baseline Compensation on History 7 5 Contrast Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 NanoPhotometer M Pearl User Manual Select Games function This determines whether the games folder is displayed or not Options
72. omassie Brilliant Blue to an unknown protein and comparing this binding to that of different known concentrations of a standard protein at 595 nm this is usually BSA bovine serum albumin The Biuret method depends on reaction between cupric ions and peptide bonds in an alkali solution resulting in the formation of a complex absorbing at 546 nm The BCA method also depends on reaction between cupric ions and peptide bonds but in addition combines this reaction with the detection of cuprous ions using bicinchoninic acid BCA giving an absorbance maximum at 562 nm The BCA process is less sensitive to the presence of detergents used to break down cell walls The Lowry method is based on the Biuret reaction Under alkaline conditions the divalent copper ion forms a complex with peptide bonds in which it is reduced to a monovalent ion Monovalent copper ion and the radical groups of tyrosine tryptophan and cysteine react with Folin reagent to produce an unstable product that becomes reduced to molybdenum tungsten blue Bound reagent changes colour from yellow to blue This binding is compared with those derived from a standard protein at 750 nm this is usually BSA bovine serum albumin Detailed protocols are supplied with these assay kits and must be closely followed to ensure accurate results are obtained A linear regression analysis of the calibration standard data points is calculated the result together with the correlation coefficient c
73. oo high Physical dilution of the sample is necessary Version 1 0 Page 10 66 IMPLEN NanoPhotometer Pearl User Manual 4 NANOVOLUME APPLICATIONS AND CUVETTE APPLICATIONS The NanoPhotometer Pearl offers a complete solution for NanoVolume and standard volume applications With the NanoPhotometer Pearl Submicroliter Cell the required sample volume ranges from 0 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 out put The NanoVolume Applications folder and the Cuvette Applications folder contain different sub folders Nucleic Acids Protein and OD 600 Cell Density Contents of these sub folders are detailed below Folder Application Recommended Measurement Cell Nucleic Acids DNA Concentration purity check and dye incorporation for DNA Submicroliter Cell Cuvette samples RNA Concentration purity check and dye incorporation for RNA Submicroliter Cell Cuvette samples Oligo Concentration purity check and dye incorporation for Oligo Submicroliter Cell Cuvette samples Protein Protein UV Protein determination at 280 nm Submicroliter Cell Cuvette Christian Warburg Protein Dye Protein determination at 280 nm and dye incorporation Submicroliter Cell Cuvette BCA Protein determination at 562 nm Cuvette Bradford Prote
74. oration 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 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
75. ows ug ml P OK Bak ug ul pmol ul mg dl mmol l umo 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 98 68 2 will 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 1 0 2 1 and 2 mm for NanoVolume applications and 5 or 10 mm for cuvette applications Step 12 Press Next to enter the Results screen OR Press Cancel Q to return to the Parameters screen Version 1 0 Page 44 66 IMPLEN NanoPhotometer Pearl User Manual Results Screen a pa a oO n 2 0 4 0 6 0 8 1 0 Ag An dA Slope ur R 0 001 0 006 O QO05 0 099 0 005 0 19557 Sample 1 Time 00 00 10 Abs 0 2276 Options select using key pad numbers Parameters Print Print DI ata Set ti At Cursor Set tn At Cursor Slope Sample Humber Save Merhad Frinter Settinas OOOOGS500
76. r Pearl directly after delivery If yes turn it of and wait 30 min before switching the unit on If both suggestions doesn t help disconnect the power completely for at least 10 seconds and then reconnect and restart the system again Remove cuvette or submicroliter cell from the cell holder turn off the instrument and start again Remove cuvette or submicroliter cell from the cell holder turn off the instrument and start again Remove the cell holder and place back in the right position If both suggestions doesn t help disconnect the power completely for at least 10 seconds and then reconnect and restart the system again Disconnect the power for quite some time to make sure that the problem is not occurring due to low energy sent to the Xenon flash lamp Disconnect the power completely for at least 10 seconds and then reconnect and restart the system again Check all connections if the cables etc are sticking right Check correct power supply 18 V is attached If possible try another power supply Please contact the Implen Support Team support implen de Phone 49 89 7263718 20 if none of the mentioned solution helps to solve the problem or if another error message should appear on the NanoPhotometer Pearl display Version 1 0 Page 60 66 IMPLEN NanoPhotometer Pearl User Manual 9 2 Trouble shooting Symptom Solution Instrument failing start up calibration Check that the correct power supply 18 V 1
77. r 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 Changing cell holder or removal for cleaning This can be removed by undoing the appropriate screws on the bottom of the instrument Version 1 0 Page 59 66 IMPLEN NanoPhotometer Pearl User Manual 9 ERROR MESSAGES AND TROUBLE SHOOTING 9 1 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 NanoPhotomete
78. rameter screen oa Version 1 0 Page 31 66 IMPLEN Calibration Screen replicates off Biuret Calibration 0 400 0 600 0 800 25 T ne 1 0 Biuret Calibration Standards 0 700 o 044 A the NanoPhotometer Pearl 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 as 0 600 0 249 A 0 800 0 349 A 0 3 1 000 D 446 4 m 1 400 0 542 A D de OK Calibration Screen replicates on Biuret Calibration Replicates 1 0 349 4 the 0 349 A mu 0 345 A UR ha OL Version 1 0 e s D4 0 6 n a LO le LM Step 13 Step 14 Step 15 Step 16 Step 17 Step 18 Step 19 Step 20 Step 21 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 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 Qo to accept the calibration and go to the Results screen see below OR Press Ba
79. rameters 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 Printer Exit options by pressing Q or wait Page 27 66 IMPLEN 4 2 6 Lowry Assay NanoPhotometer Pearl User Manual The colorimetric Lowry assay is not recommended with the Submicroliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen Lowry Parameters 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 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 keypa
80. rd 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 Qo to accept the calibration and go to the Results screen see below OR Press Back Q to cancel selections and return to the Standards screen Calibration Screen replicates on 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 Qo to display the replicate entry boxes Use C to clear previously stored results before measuring Step 20 Insert the standard and press 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 Qo to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Page 23 66 a Calibration Screen manual entry BCA Calibration 0 057 A m5 0 171 A D 288 A 0 5 t 0 404 4 E ki 0 516 A fi n3 yo 1 D B27 A A 0 2 A n a D4 D6 D B LU Ld LM E OK E Back Results screen 562 nm oUm oom Absorbance Concentration 0 255 A Curve Fit Regression
81. ressing Cancel Q Results Screen Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Insert sample and press W 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 Options to display available Options which are described below Step 13 Press Escape and confirm with Qo to return to the Functions folder Query needs confirmation to avoid unintended escaping the application folder Page 3 66 IMPLEN Options select using key pad numbers Parameters Frint Abs T Print Graph Sample Kumber Save Method Printer Settings O00 3506 Version 1 0 NanoPhotometer Pearl 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
82. right arrows Options Dilution Factor Factor are 5 mm or 10 mm BW Step 5 Enter the Dilution Factor using the keypad numbers Range 1 00 to 9 999 Use the C button to backspace Background and clear the last digit entered OR press Options to enter the dilution factor screen Enter the volume of the sample using the keypad numbers Range O O1 to 9 999 Enter the volume of the diluent using the keypad om numbers Range 0 01 to 9 999 Press 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 Step6 Background correction at 320 nm is recommended to be Step 7 Select the Units of measurement using the left and right arrows Options ug ml ng ul ug l Step 8 Enter the Factor using the keypad numbers Default BAD ee value is 50 for dsDNA 37 for ssDNA and 40 for RNA range is 0 01 to 9 999 Background Step 9 Press OK Qo to enter the Results screen OR Cancel Q to return to the Nucleic Acids folder Cuvette Applications dsD MA Parameters Results Screen Results Screen Step 10 Insert the reference sample Press Blank Key This will be used for all subsequent samples until changed A230 00534 Step 11 Insert sample and press This measures at the selected A260 DIP4A wavelengths and displays the results The sample 0 001 A concentration the ratio of A260 A280 and A260 A230 are 0 001 A calculated corrected
83. rsors 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 Printer Exit options by pressing Q or wait Page 24 66 IMPLEN 4 2 5 Bradford Assay NanoPhotometer Pearl User Manual 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 wu auelength Bradford Parameters Curve Fit Reg i Calibration 4 Standards Ld 6 ne Standards Screen Bradford Standards Std 2 Std 5 0 400 7 000 gt Ment E B ack Version 1 0 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Parameter Screen Press 2 to select Cuvette folder Press 2 to select Protein folder Press 3 to select Bradford mode The default Wavelength setting is 595 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 Pathleng
84. s 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 Press Next Qo to enter the next screen Biuret Parameters Step8 Select the Calibration mode either standards measure prepared standards manual keypad data entry or new standards using a saved method previous values are Discuss blanked new standard can be measured Step9 if standards selected Select the number of Replicates opu using the left and right arrows This determines the Standards number of standards to be measured and averaged at a oa each standard concentration point Can be OFF 1 2 or eplicates 3 Step 10 Press Next Qo to enter the Standards screen OR Press Cancel Cancel to cancel selections and return to the Protein folder Standards Screen Standards Screen Biuret Standards 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 C button backspaces and clears the last digit entered Step 12 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 Pa
85. t 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 Options key and then use the left right arrows ug ml Ug ul pmol ul mg dl mmol l umol Il 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 Step 8 Press Next Qo to enter the next screen Step9 Select the Calibration mode either standards measure prepared standards manual keypad data entry or new standards using a saved method 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 Ra
86. 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 ignores the selection pressing o accepts them Page 42 66 IMPLEN NanoPhotometer Pearl User Manual Add Peak Shortcut button 5 wavescan User Defined Peak A l i 0 30 Parameters Print Abs T Feak Detection Delete Peak Graph Scale Sample Munmber Save Method Printer Settings 0009000090 Version 1 0 Add Peak Shortcut button 5 Adds a user defined peak at the current cursor position The entry is then displayed in inverse colouring to discriminate between user defined peaks and auto detect peaks When the cursor is positioned over the user defined peak a legend User Defined Peak appears at the top of the graph The option then changes to Delete Peak to enable the user to remove the peak Graph Scale Shortcut 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 a
87. ter 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 months 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 62 66 IMPLEN NanoPhotometer Pearl User Manual 11 APPENDIX 11 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 11 2 Nucleic acid fluorescent dye incorp
88. th 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 Options key and then use the left right arrows ug ml Ug ul pmol ul mg dl mmol l umol Il 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 Qo to store the chosen parameters OR Cancel Q Press Next Qo to enter the next screen Select the Calibration mode either standards measure prepared standards manual keypad data entry or new standards using a saved method 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 Step 10 Press Next Qo to enter the Standards screen OR Press Cancel Q to cancel selections and return to the Protein folder 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
89. thod OR to return to the User Methods folder Version 1 0 Page 53 66 IMPLEN NanoPhotometer Pearl User Manual Methods Methods 1 e Single wavelength sh Delete Method i F3 Lock Method Unlock Method Delete Method 1 Select the method to be deleted using the key pad numbers 2 Select options and press 1 Delete Method 3 Press o to delete the method OR to return to User Methods folder Version 1 0 Page 54 66 IMPLEN NanoPhotometer Pearl User Manual 7 UTILITIES Survey of the available Utilities Utilities ei Select screen layout themes history and Baseline Compensation Version 1 0 Page 55 66 IMPLEN 7 1 Date and Time The procedure is as follows Date and Time Day Hour Month Minute Year 2006 gt DK amp Cancel 7 2 Regional Sets Number Format The procedure is as follows Regional Number Format 3955 3 io Cancel T 3 Printer Sets up printing options 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 M Pearl User Manual Enter the Day using the keypad numbers or left and right arrows Enter the Month as above Enter the Year Enter the Hour Enter the Minute Seconds are zeroed when OK is pressed Press OK t
90. tion too low Di 1 Concentration too high Please change to lid 50 and press sample No changes Do you want to change the lid factor automatic change of lid factor lid 10 to lid 50 in the software for calculation Concentration too low Please change to lid 10 apply a minimum of 1ul No changes Do you want to change the lid factor of sample and press sample automatic change of lid factor lid 50 to lid 10 in the software for calculation Concentration too high Dilute sample or change to lid 100 Concentration too low Please change to lid 5O 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 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 Warning message Instruction Abetoo low sample concentration is too low E sample concentration is too low Abs too low or change to lid 5 if available F Ji 50 Abs too low E J Abs is too high Physical dilution of the sample is necessary or change to lid 100 if available optional 13 2 T Abs is too high Physical dilution of the sample is necessary or change to lid 250 if available E optional L as too low E optional Abs is t
91. uring window Warning Do not overfill the well Lid Sample Pathlength Dilution Mme t m 5 optional 63 2p 100 optional 0 3 2yl 1 100 250 optional 0 3 2yl 1 250 Make sure that the lid fits exactly for the measurements 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 3 2 Software instructions 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 lid mirror Clean measurement window and lid mirror well with a slightly wet fluff free tissue Use water 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 use dry pressurized air oil free if needed Your cell is ready for the next sample Page 8 66 IMPLEN NanoPhotometer Pearl User Manual Operation Limitations Do not autoclave the unit Do not use an ultrasound bath to clean Do not drop in water or solvent bath The unit is water resistant but not water proof 3 2 Software instructions The NanoVolume Applications and Cuvette Applications are very similar concerning the analysis of dSDNA ssDNA RNA Oligonucleotides protein UV and protein dye analysis This section describes the specific features
92. usly entered calibration values and allows values to be entered via the keypad Step 22 The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9 999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Step 23 Press OK Qo to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Results screen Step 24 Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Step 25 Insert the sample and press W The concentration of the sample is taken and displayed Step 26 Repeat for all samples Step 27 Press Options to display available Options which are described below Step 28 Press and confirm with M to return to the Protein folder Query needs confirmation to avoid unintended escaping the application Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle graph on off Displays the calibration graph cursors give values for last measured sample 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
93. which have to be considered using the NanoPhotometer Pearl 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 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 oo dsD MA Parameters Step 2 Step 4 Select the Lid Factor using the left and right arrows Pathiengih 35 5 Background 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 9 66 IMPLEN NanoPhotometer Pearl 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 Mesa inewen YES LS Concentration too iow antann kt Concentration too high Please change to lid 10 and press sample No changes Do you want to change the lid factor automatic change of lid factor lid 5 to lid 10 in the software for calculation Lid 10 Concentra
94. yond the range of the instrument 2 5 A or 0 3 A then this option is greyed Out T Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Open printer settings possibility to change the printer settings within the method as described in 7 3 Printer Exit options by pressing Q or wait Page 45 66 IMPLEN NanoPhotometer Pearl 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 THERE Step3 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
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