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USER MANUAL - SERVA Electrophoresis GmbH

1. 2 Print result via selected method 4 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 Auto print toggles auto print on off Exit options by pressing or wait Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 53 Functions 5 7 Absorbance Ratio This makes simple absorbance ratio measurements on samples measuring the amount of light that has passed through a sample relative to a blank this can be air at two wavelengths The procedure is as follows Parameter Screen Absorbance Ratio wavelengths Wavelength 1 Wavelength 3 Wavelength 2 Background Absorbance Ratio Parameters Factor 7 000 Pathlength Dilution Factor 7 000 Units pg ml E Back Absorbance Ratio Parameters Yolume Diluent 0 000 ces Version 2 0 phone 49 0 89 726 3718 O Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 Step 12 Step 13 Step 14 Fax 49 0 89 726 3718 54 Page 54 Parameter Screen Press 3 to select Functions Press 7 to select Absorbance Ratio Enter the first Wavelength by using the keypad numbers or the left and r
2. Parameters Print Graph d Edit Sample Pathlenath Sample Humber Save Method Auto Frint Pana e e a aa a a a a a a a a a a a a a a a a a a a a a a a a a a a a a n a a a a a a a n a a a a n a a a a ata a n a a n a a a ala a a aa EEN Version 2 0 phone 49 0 89 726 3718 O Step 23 Step 24 Step 25 Step 26 Step 27 Step 28 Step 29 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Press OK di to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Results screen Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Insert the sample and press The concentration of the sample is taken and displayed Repeat for all samples Press Options to display available Options which are described below Press and confirm with d 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
3. Results screen Bradford Sample 1 Concentration wavelength 535 mm Absorbance 0 337 4 Curve Fit Sample Pathlength Regression Standard Pathlength 10 mm Options select using key pad numbers Farameters Frint Graph Step 22 Step 23 Step 24 Step 25 Step 26 Step 27 Step 28 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Press OK di to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Results screen Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Insert the sample and press The concentration of the sample is taken and displayed Repeat for all samples Press Options to display available Options which are described below Press and confirm with d 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
4. Wavelength range 190 1 100 nm Wavelength scan range 200 950 nm Sample volume 0 7 to 10 ul with LabelGuard Microliter Cell with cuvettes up to 3 5 ml quartz or plastic Less than 5 seconds Wavelength accuracy Stray light lt 0 5 at 220 nm using Nal and 340 nm using NaNO saturation approx ce mg ml 0 007 A BS 1 0A PE 0 005 A or 1 of the reading whichever is the gea er 0 005 A pk to pk at 0 A 260 nm Dual channel Czerny Turner with flat grating 1024 pixel CCD array concave mirrors Lamo o O Lanman S Xenon flashamoa 10 flashes up to 10 years A year S Warranty 1 year Performance verification Auto diagnostics when switched on Cell types 15 mm centre height outside dimension 12 5 mm x 12 5 mm Photometric mode Abs T concentration scan ratio multi wavelength kinetics in AAbs x factor min Built in methods Nucleid acid labelling efficiency nucleic acid protein protein and cell density Weight Input Output ports SD Memory Card USB and Bluetooth for connection to a PC for direct data download for spreadsheet calculations printout and data storage Specifications are measured after the instrument has warmed up at a constant ambient temperature and are typical of a production unit As part of our policy of continuous development we reserve the right to alter specifications without notice Note Specifications are subject to change without notice Warranty e IMPLEN
5. Wigs NanoPhotometer USER MANUAL Version 2 0 INL CN phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Implen GmbH Schatzbogen 52 D 81829 Germany Declaration of conformity for the NanoPhotometer This is to certify that the Implen NanoPhotometer conforms to the requirements of the following Directives 13 23 7EEC 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 June 1 2006 1G SOL Hn Dr Thomas Sahiri Managing Director Implen GmbH Cg phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Version 2 0 Page 2 Email info implen de www implen de Conformity Declaration TABLE OF CONTENTS 1 ESSENTIAL SARE T YN US egene ee 5 1 1 Unpacking Positioning and INSTANT ON sista cecceieeisncscesincimaannaeweneetennds cdewelccedecacenesianincadanssancawacend lt naiiandentuewanacens 5
6. 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 10 LabelGuard Microliter Cell Important Information If the absorbance value of the sample is not between 0 02 and 1 7 the following Warning message and Instruction will be displayed in the top left corner of the result screen volume i ES Sample concentration is too low optional Abs too low change to lid Abs is too high Sample concentration is too low or change to lid 5 if available change to lid change to lid Physical dilution of the sample is necessary or change to lid 100 if available change to lid Physical dilution of the sample is optional Abs is too high necessary EG phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 11 LabelGuard Microliter Cell 4 LABELGUARD APPLICATIONS AND CUVETTE APPLICATIONS The NanoPhotometer offers a complete solution for submicroliter volume and standard volume applications With the LabelGuard Microliter Cell the required sample volume ranges from O 7 ul to a maximal sample volume of 10 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 Before starting it is advisable to check that Printer Options have been appropriately set for your experiment Pl
7. E E A geed 58 T Dateand nn TEEN 59 T PR EIERE 59 o PAO EE 59 FS MA ner EEN 60 OLAS ee 60 TO POU EEN 61 MELEN phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 3 Table of contents 8 PC GES OPUS Sree eo ce sain shine E manne usin same ss E 62 9 MAINTENANCE siren E E O E E E E E 62 9 1 Maintenance tree Technology EE 62 92 Lamp Repa EMEN EE 62 9 3 Cleaning and general care of the INStrUMEnt s ssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnmnnn nnmnnn nnmnnn 62 10 SPECIFICATION AND WARRANTY cccc ccccsseeceeseeceaseecensecensesceasescaaseneaseesoaseecoasescoaseseaaseseasesensseesenseess 63 Te PIN I E 64 111 Natier acid guante at eebe 64 11 2 Nucleic acid fluorescent dye incorporati n eebe Eu 64 11 3 Protein fluorescent dye incorporation gea 66 MELEN phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 4 Table of contents 1 ESSENTIAL SAFETY NOTES There are a number of warning labels and symbols on your instrument These are there to inform you where potential danger exists or particular caution is required Before commencing installation please take time to familiarise yourself with these symbols and their meaning AN Caution refer to accompanying documents Background colour yellow symbol and outline black 1 1 Unpacking Positioning and Installation e Check t
8. 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 I g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed Decimal Points DP to be selected from O to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98 768 2 will display as 98768 even with 1 decimal point selected Press OK d to store the chosen parameters OR Cancel ve Press Next d to enter the next screen Biuret Parameters Step 8 Select the Calibration mode either standards measure prepared standards manual keypad data entry or new standards using a saved method previous values are Regression blanked new standard can be measured GE Step 9 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 e each standard concentration point Can be OFF 1 2 or et i Step 10 Press Next d to enter the Standards screen OR Press Cancel to cancel selections and return to the Protein folder Standards Screen Standards Screen Bare Sandane Step 11 Enter the concentration values by using the keypad numbers and the up and down arrows to move between
9. Toggle graph on off Displays the calibration graph cursors give values for last measured sample 4 Possibility to edit the sample pathlength 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 Auto print toggles auto print on off Exit options by pressing or wait Fax 49 0 89 726 3718 54 Page 27 Email info implen de www implen de LabelGuard Cuvette Applications 4 2 5 Bradford Assay The colorimetric Bradford assay is not recommended with the LabelGuard Microliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen wavelength Units Bradford Parameters Pathlength ze Bradford Parameters Bradford Parameters Regression Calibration Standards Standards Screen Bradford Standards Std 1 Std 4 Std f Std 3 Std 5 0 400 1 000 Std 3 Std 6 0 600 1 400 e em EEN Version 2 0 phone 49 0 89 726 3718 O 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 3 to select Bradford mode The default Wavelength setting is 595 nm Enter the number
10. use the down arrow Sample 1 A 450nm 465 0 164 A Step 11 Press Options to display available Options which are described next Step 12 Press and confirm with d to return to the Functions folder Query needs confirmation to avoid unintended escaping the application INL EN phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 44 Functions Options select using key pad numbers Faraneters Print Abst SI Peak Detection Add Peak Graph Scale Sample Mumber Save Method Auto Frint 668606006 Peak Detection Shortcut button 4 Peak Detection Auto detect Peaks Peak Detect on Zoom Hin Pk Height Sort Peaks By aa 11 enen Hin Pk width Oraw Peaks me IIe Dr Dk amp Cancel wW avescan 0 20 L 0 25 A 0 20 H 0 15 E i 3 oo DI A e E Een Lk seen mm mm mm ch 400 470 440 den 480 Son Cace e e LI Abs Jusduangdosdoal Sample 1 A 450nm 465 0 164 4 Ce phone 49 0 89 726 3718 O Version 2 0 Fax 49 0 89 726 3718 54 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 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 t
11. 2 INTRODUCTION EE 6 SN S de EE e OLN E 6 e Sami Ole Wal ONS Ns E 6 SC WEY OA ANG En En EE D 3 THE LABELGUARD MICROLITER CELL u cccsccssssssessscssessssessseesessscesessesevsscesassecevarsesavsesevassesevseaveseesarens 9 3 1 Technical d ei te EE H 3 2 Software ln Eer da E 10 4 LABELGUARD APPLICATIONS AND CUVETTE APPLICATIONS oe cceceeceeceeceeceeceeeeeeeseeseeseuseeseeeeeess 12 4 1 Characterization of DNA RNA and Oligonucleotides ceceeeeeeceeeeeeeeeeeseneeeseaseaseeeeneeassassasseeseeseeeneeneeneenees 12 ALL TCE TAIT ON Ne UO Ml EE 12 4 1 2 Analysis of dsDNA SSDNA andHNA 14 4 1 3 Analysis e glewen TEE 16 4 1 4 Dye incorporation for dsDNA SSDNA RNA and Oligonucleotides ccecceceeceeceeteeeeseeteeseeeeeeesaees 18 4 2 Protein Determination E 20 421 General Tage e WEE 20 Ee am POEMU DEE EN A235 Protein UV Dye Method E 23 E e chs EE 25 E Badon E See ne eee eee tre ee eee ee Cee ee ee ee ere ee 28 Nee E 31 e AUT ASSAY EE 34 4 3 Bacterial Cell Culture Measurement ODGOO cccesenseneeeeesenscnseeensensensesensessnsenscnssensensenensensensenensenes 37 4 3 1 General Information EE 3 4 3 2 Analysis of Bacterial Growth EE 38 5 silCleantel LN 39 5S1 Snelle Wavelength ADS and 760 E 40 52 OPC STU UI ON EG 42 53 AVES EEN 44 FUNC e 47 e ME Ce E ge ber TEE 49 BO Multiple Wavelengths a aaa aaa a aa 52 S ASDI e RANO EEN 54 6 USER MEINOD S aoia EE 56 7 EE gpi
12. 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 Ce phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 13 LabelGuard Cuvette Applications 4 1 2 Analysis of dsDNA ssDNA and RNA The procedure is as follows Parameter Screen Parameter Screen Step 1 Press 1 for LabelGuard OR 2 for Cuvette folder LabelGuard 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 LabelGuard Applications select the Lid Factor as described under 3 2 Using Cuvette Applications select Pathlength using the left and right Dilution Factor Factor arrows Options are 5 mm or 10 mm Step 5 Enter the Dilution Factor using the keypad numbers Range 1 00 to 9999 Use the C button to backspace and Background 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 9999 Enter the volume of the diluent using the keypad numbers R
13. E Results screen wavelength r50 mm Absorbance 0 055 4 Concentration 0 627 Curve Fit Regression Sample Pathlength Standard Pathlength 10 mm Options select using key pad numbers Parameters Print Graph d Edit Sample Pathlenath Sample Humber Save Method Auto Frint Para a e e aa a a a a a a a a a a a a a a a a a a a a a a a a a a a a a n a a a a a a a n a a a a n a a a a a a a n a a a a a a ala a a aa EEN Version 2 0 phone 49 0 89 726 3718 O Step 22 Step 23 Step 24 Step 25 Step 26 Step 27 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Press OK di to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Results screen Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Insert the sample and press The concentration of the sample is taken and displayed Repeat for all samples Press Options to display available Options which are described below Press and confirm with di to return t
14. Enter the volume of the diluent using the keypad numbers Range 0 01 to 9999 Press d to calculate the dilution factor and return to the Parameters screen OR Press Cancel to cancel the selections and return to the Parameters screen 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 Enter A280 Factor using the keypad numbers Default value is 1 55 Range is 1 00 to 9999 Select the Units of measurement using the left and right arrows Options mg ml ug ml ng ul and ug ul Enter the protein dependent extinction coefficient Range is 10000 to 9999999 Press OK to store the chosen parameters and to enter the next screen OR Cancel to return to the Protein folder 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 coefficient and dye dependent correction factor at 280 nm 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 3 Protein fluorescent dye incorporation Range are Dye Abs Max 190 nm to 1100 nm Dye Ext Coefficient 10000 to 9999999 Dye Correction 0 001 to 0 999 Email info implen de ww
15. 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 e Background correction at a wavelength totally separate from the nucleic acid and protein peaks at 260 and 280 nm respectively is sometimes used to compensate for the effects of background absorbance The wavelength used is 320 nm and it can allow for the effects of turbidity high absorbance buffer solution and the use of reduced aperture cells The instrument can use background correction e lf it is used there will be different results from those when unused because Abs320 is subtracted from Abs260 and Abs280 prior to use in equations Concentration Abs 260 Abs 320 Factor Abs ratio Abs 260 Abs 320 Abs 280 Abs 320 Abs ratio Abs 260 Abs 320 Abs 230 Abs 320 e lf your laboratory has not used background correction before set this option to NO e The use of background correction can remove variability due to handling effects of low volume disposable cells Spectral scan of nucleic acid Pure Nucleic Acid Poly dAdT 3 gt N Wave 260 0 Abs 0 567 gt O L CO oa L Wave 280 0 Abs 0 409 Absorbance A CO N 2 Sr 3 CH 210 0 260 0 310 0 360 0 410 0 Wavelength nm Note e absorbance maximum near
16. Press 1 to select Nucleic Acids folder Factor Step 3 Press 4 to select Oligo mode mom Step 4 Using the LabelGuard Applications select the Lid Factor as described under 3 2 Using Cuvette we mm Factor Applications select Pathlength using the left and right mmm ooo arrows Options are 5 mm or 10 mm Step 5 Enter the Dilution Factor using the keypad numbers Range 1 00 to 9999 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 9999 Enter the volume of the diluent using the keypad numbers Range 0 01 to 9999 Press to calculate the dilution factor and return to the Parameters screen OR Press Cancel to cancel the selections and return to the e eRe E i Parameters screen Step 6 Background correction at 320 nm is recommended to be eas switched on Step 7 Select the Units of measurement using the left and right arrows Options ug ml ng ul ug ul and pmol ul o O O O Step 8 Enter the Factor using the keypad numbers Default z value is 33 range is 0 01 to 9999 a Sa Step 9 If pmol l is selected there are two options to set the factor 1 A selection table denoting the ratios of the 4 bases e 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 9999 2 Enter the
17. 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 Gesi Eech DEEL foe ens A W F a a Methods 7 SE EL Cox s EEN EEN C a Methods 4 E a l Methods 9 fo ens Folder names can be renamed locked unlocked and saved to the SD memory card using the Options menu Options select using key pad numbers 19 Folder Names d Lock Folder E Unlock Folder C SO Merman Card Rename Folder Names 1 Press 1 to select Folder Names 2 Select the method to be renamed using the left and right arrows 3 Enter the new name 4 Press d to save the new name OR Q 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 Selecta pass code using the keypad numbers or left and right arrows 4 Press di to lock the method OR 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 gt to unlock the method OR 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 de
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 Insert the standard use C to clear previously stored results before measuring and press d 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 input Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Press OK d 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 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Press d 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 d to accept the calibration and go to the Results screen see below OR Press Back
19. a a a a a a a a n n n n n n n n n n a INEL CN phone 49 0 89 726 3718 0 Version 2 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 B and confirm with d 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
20. are blanked new standard can be measured if standards selected Select the number of standards to be measured and averaged at each standard concentration point Can be OFF 1 2 or 3 Press Next d to enter the Standards screen OR Press Cancel to cancel selections and return to the Functions folder Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 49 Functions Standard Screen Standard Curve Standards Std 1 Std 4 Std 2 Std 5 CR Std 3 oe Calibration Screen replicates off Standard Curve Calibration 245 Standard Curve Calibration d Standards 10 0 0 015 4 0 15 i 20 0 0 049 4 J d 30 0 0 082 A a 0 10 BS 40 0 0 115 A ae Da 50 0 0 147 4 a 60 0 0 1795 A 0 05 DE 0 05 DN Step 20 Step 22 Step 23 Version 2 0 Page 50 phone 49 0 89 726 3718 O Step 11 Step 12 Step 13 Step 14 Step 15 Step 16 yo Step 17 Step 18 Step 19 Step 21 Fax 49 0 89 726 3718 54 Standards screen Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range 0 001 to 9999 Press Next to enter the Calibration screen If any duplicate or non monotonic increasing entries are present the unit will beep and highlight the incorrect entry OR Press Back to return to the Parameter screen Calibration Screen replicates off
21. di to accept the calibration and go to the Results screen see below OR Press Back to cancel selections and return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Press d to display the replicate entry boxes Use C to clear previously stored results before measuring Insert the standard and press d 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 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 Press d to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 26 LabelGuard Cuvette Applications Calibration Screen manual entry BCA Calibration 0 051 4 7 DP 0 171 A 0 288 A d 0 404 A E pi 0 516 A ra 0 4 p d 0 627 A At D p d K Me DH 0 6 CR LH Lg LH De OK amp Back Results screen wW avelength FB ppm Absorbance 0 255 4 Curve Fit Sample Pathlength Regression Standard Pathlength 10 mm Options select using key pad numbers
22. line on and off Note if any data points enclosed by to and tn are beyond the range of the instrument gt 2 5 A or lt 0 3 A then this option is greyed out 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 Auto print toggles auto print on off Exit options by pressing A or wait Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 48 Functions 5 5 Standard Curve The construction of a multi point calibration curve from standards of known concentration to quantify unknown samples is a fundamental use of a spectrophotometer this instrument has the advantage of being able to store this curve as a method using up to 9 standards To include a zero concentration standard include this in the number of standards to be entered and enter 0 00 for concentration use a reagent blank when required to enter the zero standard The procedure is as follows Parameter Screen Standard Curve Parameters wavelength Pathlength Units de Ment E Cancel Standard Curve Parameters Standard Curve Parameters Curve Fit ae Regression a Calibration Standards Replicates ih gt Ment EA Cancel Cg phone 49 0 89 726 3718 O Version 2 0 Step 1 Step 2 Step 3 Step
23. of Standard concentration points 1 9 to be used in the curve using the keypad numbers or left and right arrows Select Pathlength using the left and right arrows Options are 5 or 10 mm Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Options key and then use the left right arrows ug ml ug ul pmol ul mg dl mmol l mol 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 d to store the chosen parameters OR Cancel Press Next d 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 gt to enter the Standards screen OR Press Cancel to cancel selections and return to the Protein folder Standards Screen Enter the concen
24. or plastic cells e The optical height of the NanoPhotometer is 15 mm e The minimum volume that can be used is 0 7 ul with the LabelGuard Microliter Cell e 12mm test tubes may be used e g for cell cultures however they are not recommended as higher quality data is produced by using disposable cuvettes for the analysis If used align the indicator line on 12 mm test tubes in the same direction to ensure reproducible positioning of the tube Note that test tubes do not last forever and that the surface becomes scratched and blemished through repetitive use if this is the case they should be replaced MELEN phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 6 Introduction 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 LCD Display On off key e We A Alphanumeric keys DOE pog Cellholder e Ca Escape Cancel Back Arrow keys aa A Dee e N Blank Reference View options Sample Enter selection OK 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 l common to all applications and described below Options Vi
25. the absorbance at 600 nm known as OD 600 reaches approximately 0 4 prior to induction or harvesting A linear relationship exists between cell number density and OD 600 up to approx 0 6 Stationary Decline Log Phase 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 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 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 Software The use of 10 mm pathlength disposable cells is recommended f
26. to return to the Standards screen Email info implen de www implen de Functions Calibration Manual entry Standard Curve Calibration c C Ke e D wy E Results screen Standard Curve Options select using key pad numbers Parameters Print Graph Edit Sample Pathlenath Sample Murober Save Method Subo Print ENEE n a n n a a n a a n n a a n a a n a a a a n a a a a a a n a a n a a a a a a n a a n a a a a a a ala ata a a a ces Version 2 0 phone 49 0 89 726 3718 O Step 24 Step 25 Step 26 Step 27 Step 28 Step 29 Step 30 Step 31 Calibration Manual entry Shows previously entered calibration values and allows values to be entered via the keypad The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Press OK di to accept the calibration and go to the Results screen See below OR Press Back to return to the Standards screen Results screen Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Insert the sample and press The concentration of the sample is taken and displayed Repeat for all samples Press Options to display available Options which are described below Press and conf
27. tyrosine tryptophan and cysteine react with Folin reagent to produce an unstable product that becomes reduced to molyobdenum tungsten blue Bound reagent changes colour from yellow to blue This binding is compared with those derived from a standard protein at 750 nm this is usually BSA bovine serum albumin Detailed protocols are supplied with these assay kits and must be closely followed to ensure accurate results are obtained A linear regression analysis of the calibration standard data points is calculated the result together with the correlation coefficient can be printed out A correlation coefficient of between 0 95 and 1 00 indicates a good straight line Ce phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 20 LabelGuard Cuvette Applications 4 2 2 Protein UV Method The procedure is as follows Parameter Screen LabelGuard Applications Protein U Parameters Parameter Screen Step 1 Press 1 for LabelGuard OR 2 for Cuvette folder Lid Factor A260 Factor Step 2 Press 2 to select Protein folder Step 3 Press 1 to select Protein UV mode Step 4 Using LabelGuard Applications select the Lid Factor as Dilution Factor A280 Factor described under 3 2 A minimum of 2 ul sample volume is recommended Using Cuvette Applications select Pathlength using the left and right arrows Options are 5 Background Units mm or 10 mm SS aes Step 5 Enter the Dilution Factor
28. 18 0 Version 2 0 Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle graph on off The graph shows a wavescan plot across the range 220 nm to 750 nm with cursors denoting 230 260 280 and if background correction selected 320 nm 4 Toggle on off to display the data table only in the print out 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Auto print toggles auto print on off Exit options by pressing OR wait Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 17 LabelGuard Cuvette Applications 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 or Analysis of Oligonucleotides To determine the dye incorporation rate the absorbance reading at the wavelength reported for maximum absorbance of the fluorescence dye is used For further details please refer to 11 2 Nucleic acid fluorescent dye incorporation The procedure is as follows Parameter Scree
29. 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 Step 5 Select the Mode Absorbance or Transmission using erem the left and right arrows Step 6 Select the Pathlength using the left and right arrows Options are 0 1 0 2 1 and 2 mm for LabelGuard applications and 5 or 10 mm for cuvette applications End Wavelength FOO nm gt Dk e Cancel ae Step 7 To enter the measurements screen with the selected parameters press OK di OR cancel the selections and return to the Functions folder by pressing Cancel Q Measurement Screen SE Measurement Screen Step 8 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 9 Insert sample and press d Step 10 Repeat for all samples Sample A 450nm Results Screen Results Screen BC i A graph of the wavescan is displayed along with a table of Absorbance T at each peak Up to eight peaks can i be shown Use the left and right arrows to move the P eo bt cursor along the graph When it reaches a peak the peak bi ped A height and width of the peak is displayed at the top of 0 10 me ness the screen To zoom in on the wavelength scale use the up arrow This auto scales on the Absorbance T scale dependent on the Graph Scale option and this is retained for subsequent measurements To zoom out again
30. 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Parameter Screen Press 3 to select Functions Press 5 to select Standard Curve Select the Wavelength using the keypad numbers or left and right arrows Enter the number of Standard concentration points to be used in the curve 1 9 Select the Pathlength using the left and right arrows Options are 0 1 0 2 1 and 2 mm for LabelGuard applications and 5 or 10 mm for cuvette applications 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 I g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed Decimal Points DP to be selected from O to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98 768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters or Cancel Select the type of Curve Fit using the left and right arrows Options straight line regression a zero regression this forces the straight line through the origin interpolated or cubic spline Select the Calibration mode either Standards measure prepared standards or Manual keypad data entry or new standards using a saved method previous values
31. Concentration with correction of dye contribution to the 260 nm absorbance reading Cnuc A260 CFaye Amax dye Factornuc Lid factor Dilution factor Cnuc nucleic acid concentration ng ul A260 absorbance AU of nucleic acids CFaye dye dependent correction factor at 260 nm Amax dye absorbance at absorption maximum of the dye AU Factornuc 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 or 100 Dependent on the used LabelGuard Lid e Calculation of the Dye concentration Cdye Amax dye Lid factor Dilution factor Edye 10exp 6 Cdye dye concentration pmol ul Amax dye absorbance at absorption maximum of the dye AU Lid Factor virtual dilution factor 5 10 50 or 100 Dependent on the used LabelGuard Lid Edye dye dependent extinction coefficient M cm t e Calculation of the Frequency of Incorporation FOI of dye per 1 000 bases Formula for dsDNA FOI 6 49 Amax dye Edye 10exp 6 A260 corrected Formula for ssDNA FOI 8 77 Amax dye Edye 10exp 6 A260 corrected Formula for RNA FOI 8 11 Amax dye Edye LOexp 6 A260 corrected Formula for Oligonucleotides FOI 9 83 Amax dye Edye LOexp 6 A260 corrected Amax dye absorbance at absorption maximum of the dye AU Edye dye dependent extinction coefficient M cm t A260 corrected A260 CFaye Amax dye INL CN ph
32. N phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 39 Functions 5 1 Single Wavelength Abs and T 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 Parameter Screen Single Wavelength Parameters Step 1 Press 3 to select Functions Step 2 Press 1 to select Single Wavelength KE Step 3 Set Wavelength by using keypad numbers or left and right arrows Step 4 Select the Mode Absorbance or Transmission using the left and right arrows Step 5 Select the Pathlength using the left and right arrows E Options are 0 1 0 2 1 and 2 mm for LabelGuard applications and 5 or 10 mm for cuvette applications Step 6 To enter the results screen with the selected parameters Lo o EN Cancel press OK d OR cancel the selections and return to the Functions folder by pressing Cancel Q Results Screen Single wavelength Results Screen Step 7 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 8 Insert sample and press W 450 nm Step 9 Repeat for all samples 150 100 Js Li a ER ER a LU a m a a 0 Units KA Step 10 The result at the selected wavelength is displayed on the screen Step 11 Use t
33. O to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98 768 2 will display as 98768 even with 1 decimal point selected Press OK d to store the chosen parameters or Cancel Step 8 Select the Pathlength using the left and right arrows Options are 0 1 0 2 1 and 2 mm for LabelGuard applications and 5 or 10 mm for cuvette applications Step 9 To enter the results screen with the selected parameters press d OR cancel the selections and return to the Functions folder by pressing Cancel GA Results Screen if using a Factor Concentration Results Screen if using a Factor Step 10 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Step 11 Insert sample and press wi EG phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 42 Functions Results Screen if using standard mode Concentration Run Standard Concentration Concentration Options select using key pad numbers Parameters Frint Graph Fun Standard Sample Mumber Save Method 4 uto Frint INL CN phone 49 0 89 726 3718 0 Version 2 0 Fax 49 0 89 726 3718 54 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 d
34. aaa the different standard boxes Range 0 001 to 9999 C button backspaces and clears the last digit entered Std 5 Step 12 Press Next d 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 sun SEA Back to return to the Parameter screen EG phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 34 LabelGuard Cuvette Applications Calibration Screen replicates off Biuret Calibration Standards 0 200 0 400 0 600 0 800 d DH DR DR 40 Jg L4 Biuret Calibration Standards A 0 200 0 044 4 d Sg 0 400 0148A i 0 600 0 800 1 000 0 243 A 0 3454 ma 0 446 4 ei 1 400 ba 0 542 4 x D L Gei Iw DH OE DR 10 Le LH 2 Back Calibration Screen replicates on Biuret Calibration Replicates a 1 0 249 4 the H2 0 349 A E Da sl 0349A 1 af m K 0 300 ns DH OE DR LO Le LH Version 2 0 phone 49 0 89 726 3718 O Step 13 Step 14 Step 15 Step 16 Step 17 Step 18 Step 19 Step 20 Step 21 Fax 49 0 89 726 3718 54 Page 35 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
35. acid fluorescent dye incorporation 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 between 0 02 and 1 7 a Warning message and Instruction will be displayed in the top left corner of the result screen Please refer to 3 2 Software instructions for further information Repeat for all samples Press Options to display available Options which are described below Press and confirm with gt to return to the Nucleic Acids folder Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 18 LabelGuard Cuvette Applications Options select using key pad numbers Farameters Frint Graph Frint Data Only Sample Number Save Method Auto Frint INL CN phone 49 0 89 726 3718 0 Version 2 0 Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle graph on off The graph shows a wavescan plot across the range 220 nm to 750 nm with cursors denoting 230 260 280 and if background correction selected 320 nm 4 Toggle on off to display the data table only in the pr
36. aken and displayed Repeat for all samples Press Options to display available Options which are described below Press and confirm with d 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 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 Auto print toggles auto print on off Exit options by pressing Q or wait Fax 49 0 89 726 3718 54 Page 36 Email info implen de www implen de LabelGuard Cuvette Applications 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
37. ange 0 01 to 9999 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 z Step 6 Background correction at 320 nm is recommended to be Pathlength Units ii Step 7 Select the Units of measurement using the left and right ee z arrows Options ug ml ng ul ug ul Step 8 Enter the Factor using the keypad numbers Default mn O value is 50 for dsDNA 37 for ssDNA and 40 for RNA Ge p range is 0 01 to 9999 aCKOroOUn Step 9 Press OK d to enter the Results screen OR Cancel Q to return to the Nucleic Acids folder d OK E Cancel Cuvette Applications dsO0HNHA 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 oss Step 11 Insert sample and press This measures at the selected wavelengths and displays the results The sample concentration the ratio of A260 A280 and A260 A230 are calculated corrected by the background wavelength value if selected Ee Step 12 If the absorbance value of the sample is not between ee ee 567 0 02 and 1 7 a Warning message and Instruction will be displayed in the top left corner of the result screen 0 AZBOA230 Please refer to 3 2 Software instructions for further 156 566 information Step 13 Repeat for all samples Step 14 Press Options to display availabl
38. ation 0 044 4 0 146 4 the gt 0 2495 A E Ki 0 349 4 D 0 446 A d 0 542 4 P DU D EI H i l Pad CS DH D E WR log lE LH o ok i Back Results screen Some 1 Concentration wW avelength B46 nm Absorbance 0 249 4 Curve Fit Sample Pathlength Regression Standard Pathlength 10 mm Options select using key pad numbers Parameters Print Graph d Edit Sample Pathlensth Sample Humber Save Method Subo Frint ENNEN aa aa aaa a aaa a aaa aaa a aaa a a aa a a aa a a a a a a a a a a a a a a a a a a aia ea EEN Version 2 0 phone 49 0 89 726 3718 O Step 22 Step 23 Step 24 Step 25 Step 26 Step 27 Step 28 Calibration Screen manual entry Shows previously entered calibration values and allows values to be entered via the keypad The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Press OK di to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Results screen Insert the reference sample and press Blank key This will be used for all subsequent samples until changed Insert the sample and press The concentration of the sample is t
39. be applied to other proteins if the corresponding factors are known please note that the factor used by the NanoPhotometer is the reciprocal value of the extinction coefficient 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 To customize the equation for a particular protein the absorbance values at 260 and 280 nm should be determined at known protein concentrations to generate simple simultaneous equations solving these provides the two coefficients In cases where A260 Factor is found to be negative it should be set to zero since it means there is no contribution to the protein concentration due to absorbance at 260 nm Set A260 Factor 0 00 for direct A280 UV protein measurement A280 Factor is based on the extinction coefficient of the protein molecular weight molar extinction coefficient If BSA bovine serum albumin is an acceptable standard setting A280 Factor 1 588 will give linear results from 0 1 to 160 mg ml protein Protein mg ml 1 588 Abs 280 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 LabelGuard Applications and Cuvette Applications To determine the degree of labellin
40. calibration graph cursors give values for last measured sample Edit Sample Pathlength 4 Possibility to edit the sample pathlength 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 Auto print toggles auto print on off Exit options by pressing Q or wait Sample Humber Save Method Auto Frint Manaa aaea a aaa a aa a a a a a a a aa a a aa a a aa a a a a a a aa a a aa a a a aa a a aa a a a a a a aa a a aa a a a a aaa a EEN phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 30 LabelGuard Cuvette Applications 4 2 6 Lowry Assay The colorimetric Lowry assay is not recommended with the LabelGuard Microliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen Parameter Screen Step 1 Press 2 to select Cuvette folder Step 2 Press 2 to select Protein folder elek Step 3 Press 4 to select Lowry mode Step 4 The default Wavelength setting is 750 nm Step 5 Enter the number of Standard concentration points 1 9 to be used in the curve using the keypad numbers or left ps and right arrows Step 6 Select Pathlength using the left and right arrows Options are 5 or 10 mm Lowry Parameters Step 7 Units T
41. 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 gt 1 8 and 2 0 respectively deviations from this indicate the presence of impurity in the sample but care must be taken in interpretation of results The 260 nm reading is taken near the top of a broad peak in the absorbance spectrum for nucleic acids whereas the 280 nm reading is taken on a steep slope i e small changes in wavelength cause large changes in absorbance Consequently small variations in wavelength at 280 nm will have a greater effect on the 260 280 ratio than variations will at 260 nm Thus different instruments of the same and different types may give slightly different ratios due to variations in wavelength accuracy But each instrument will give consistent results within itself Concentration also affects 260 280 readi
42. e Options which are described below Step 15 Press and confirm with d to return to the Nucleic Acids folder EG phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 14 LabelGuard Cuvette Applications Options select using key pad numbers Farameters Frint Graph Frint Data Only Sample Number Save Method Auto Frint INL CN phone 49 0 89 726 3718 0 Version 2 0 Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle graph on off The graph shows a wavescan plot across the range 220 nm to 750 nm with cursors denoting 230 260 280 and if background correction selected 320 nm 4 Toggle on off to display the data table only in the print out 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Auto print toggles auto print on off Exit options by pressing OR wait Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 15 LabelGuard Cuvette Applications 4 1 3 Analysis of Oligonucleotides The procedure is as follows Parameter Screen LabelGuard Applications Parameter Screen Oligo Parameters Step 1 Press 1 for LabelGuard OR 2 for Cuvette folder Step 2
43. ease 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 MELEN phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 5 Essential Safety Notes 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 NanoPhotometer IN ss ney pag Description d LabelGuard number Life Science methods such as nucleic acid assays and protein assays using the LabelGuard Microliter Cell 2 Life Science methods such as nucleic acid assays protein assays and cell density Applications K Luuette 4 SPplic ations Functions Contains nine folders that can store user adapted 3 General spectroscopic methods Si User Me
44. ease refer to 7 3 Printer The LabelGuard 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 samples LabelGuard Cuvette RNA Concentration purity check and dye incorporation for RNA samples LabelGuard Cuvette Oligo Concentration purity check and dye incorporation for oligo samples LabelGuard Cuvette Protein Protein UV Protein determination at 280 nm LabelGuard Cuvette Christian Warburg Protein Dye Protein determination at 280 nm and dye incorporation LabelGuard Cuvette BCA Protein determination at 562 nm Cuvette Bradford Protein determination at 595 nm Cuvette Lowry Protein determination at 750 nm Cuvette Biuret Protein determination at 546 nm Cuvette Cell Count OD600 Cell density at 600 nm Cuvette 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
45. entration pmol wul Caye Amax dye Lid factor Dilution factor aye 10exp 6 Caye dye concentration pmol ul Amax dye absorbance at absorption maximum of the dye AU Lid factor virtual dilution factor 5 10 50 or 100 Dependent on the used LabelGuard Lid Edye dye dependent extinction coefficient M cm t e Calculation of Degree of Labelling D P Degree of Labelling Amax aye Eprot A280 Amax aye CFaye Edye Amax dye absorbance at absorption maximum of the dye AU Eprot protein dependent extinction coefficient M cmt CFaye dye dependent correction factor at 280 nm Edye dye dependent extinction coefficient M cmt The following dye types and parameters are pre programmed in the NanoPhotometer Dye dependent extinction Dye dependent correction Absorption maximum coefficient factor 280 nm Dye Type Dyes nm Edye CF bye 346 19000 401 34500 495 71000 650 239000 550 150000 649 250000 654 250000 493 70000 495 68000 416 46000 566 200000 595 80000 In all formulas the molar dye dependent and the molar protein dependent extinction coefficient is used MELEN phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 66 Appendix
46. es and clears the last digit entered Press Next d to enter the Calibration screen If there are duplicate or non monotonic increasing entries the unit will beep and highlight the incorrect entry OR Press Back to return to the Parameter screen Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 25 LabelGuard Cuvette Applications Calibration Screen replicates off BCA Calibration d DH DR DR 40 Jg L4 ECA Calibration 1 000 0 516 4 1 400 ouezes E ai bk no DH OE DR LO Le LH o Replicates A 1 D626 4 E 0 626 4 ha oR 0 626 A De g uf d DL oz DH DR DR 4140 tLe Lu Ss MALEN phone 49 0 89 726 3718 O Version 2 0 Step 14 Step 15 Step 16 Step 17 Step 18 Step 19 Step 20 Step 21 Step 22 Calibration Screen replicates off This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Insert the standard use C to clear previously stored results before measuring Press 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
47. eters 1 Step 1 Step 2 Wavelength Delay Time Step 3 Seconds Step 4 Duration 1 Minute Step 5 Interval 10 Seconds a gt Meu E Cancel Step 6 Step 7 Kinetics Parameters 2 Step 8 Mode Pathlength Factor e E DE i Back Kinetics Parameters 7 Step 10 Step 11 Step 12 Parameter Screen Press 3 to select Functions Press 4 to select Kinetics Wavelength Enter all numerical values using the keypad numbers or the left and right arrows Delay time Enter the delay time in seconds before the first measurement is taken This can be a maximum of 600 seconds 10 minutes Duration Enter the time in minutes over which measurements are taken This can be a maximum of 60 minutes Interval Enter the interval time in seconds between measurements using the left and right arrows Options are 5 10 20 30 or 60 seconds Press Next to go to the next parameters screen OR Press Cancel to return to the Functions folder 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 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 arr
48. ew Options l n l l 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 Escape from a selection and return to the previous folder Set reference to 0 000 A or 100 T 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 Back o Enter or confirm a selection Take a measurement Alphanumeric keys MELEN phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page Introduction Options select using key pad numbers Options select using key pad numbers Boe ee 1 View parameters for the experiments Print 2 Print the results Sech 3 4 Is described in 4 LabelGuard Applications and Frint Data Only Cuvette Applications T Define the sample number you wish to start from 8 Save the parameters as a method to a defined folder Sample Number name with a defined method name Save Method 9 Toggle auto print on off Default is off Subo Frint Exit options by pressing Escape Q or wait Experienced operators can use the numeric keys as a shortcut to the option required without need
49. g 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 3 Protein fluorescent dye incorporation Colorimetric Bradford Biuret BCA and Lowry protein determination Cuvette Applications The Bradford method depends on quantifying the binding of a dye Coomassie Brilliant Blue to an unknown protein and comparing this binding to that of different known concentrations of a standard protein at 595 nm this is usually BSA bovine serum albumin The Biuret method depends on reaction between cupric ions and peptide bonds in an alkali solution resulting in the formation of a complex absorbing at 546 nm The BCA method also depends on reaction between cupric ions and peptide bonds but in addition combines this reaction with the detection of cuprous ions using bicinchoninic acid BCA giving an absorbance maximum at 562 nm The BCA process is less sensitive to the presence of detergents used to break down cell walls The Lowry method is based on the Biuret reaction Under alkaline conditions the divalent copper ion forms a complex with peptide bonds in which it is reduced to a monovalent ion Monovalent copper ion and the radical groups of
50. 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 Ce phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 63 Specification and warranty 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 Cnuc A260 Factornuc Lid Factor Cnuc nucleic acid concentration ng ul A260 absorbance AU of nucleic acids Factornuc 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 or 100 Dependent on the used LabelGuard Lid maximum absorbance wavelength dye dependent correction factor at 260 nm dye dependent extinction coefficient 11 2 Nucleic acid fluorescent dye incorporation To determine the nucleic acid concentration and the dye concentration after probe labelling a modification of the Lambert Beer equation is used e Calculation of the Nucleic Acid
51. h 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 di to accept the calibration and go to the Results screen see below OR Press Back to cancel selections and return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Press d 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 d to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Email info implen de www implen de LabelGuard Cuvette Applications Calibration Screen manual entry Lowry Calibration UU A 0 056 A 0 094 A T i yi 0 132 4 ES 0 169 A 0 205 A Ki a Dei DH Dh DR Lo Lg LH 2 Back a Ke
52. he NanoPhotometer the value is 15 mm 3 1 Technical instructions Step 1 Insert the LabelGuard Microliter 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 blue arrows Insert the LabelGuard Microliter Cell always in the same direction Pipette the appropriate sample volume onto the centre of the measuring window Warning Do not overfill the well Lid Sample Pathlength Dilution volume 100 optional 0 7 4 ul 1 100 0 7 4 ul 3 5ul 5 optional 6 10 ul 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 lid off and retrieve 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 fluff free tip or laboratory wipe 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 phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www im
53. he contents of the pack against the packing list If any shortages are discovered inform your supplier immediately e Inspect the instrument for any signs of damage caused in transit If any damage is discovered inform your supplier immediately e Ensure your proposed installation site conforms to the environmental conditions for safe operation Indoor use only Temperature range 5 C to 35 C Note that if you use the instrument in a room subjected to extremes of temperature change during the day it may be necessary to recalibrate by switching off and then on again once thermal equilibrium has been established 2 3 hours Maximum relative humidity of 80 up to 31 C decreasing linearly to 50 at 40 C e The instrument must be placed on a stable level bench or table that can take its weight lt 4 5 kg so that air can circulate freely around the instrument e This equipment must be connected to the power supply with the power cord supplied It can be used on 90 240 V 50 60 Hz supplies e lf the instrument has just been unpacked or has been stored in a cold environment it should be allowed to come to thermal equilibrium for 2 3 hours in the laboratory before switching This will prevent calibration failure as a result of internal condensation e Switch on the instrument via the keypad after it has been plugged in The instrument will perform a series of self diagnostic checks e Please read through this user manual prior to use e Pl
54. he left and right arrows to move the cursor and A display the value at the cursor position 15nm from i S set wavelength i pes Step 12 Press Options to display available Options which are eel described below A pe 0 S 164 Step 13 Press Escape and confirm with D to return to the A Functions folder Query needs confirmation to avoid oe Waite ne unintended escaping the application folder Single wavelength 440 4 EG phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 40 Functions Options select using key pad numbers Parameters Frimt Abs T Print Graph Sample Mumber Save Method Awuto Print INEL CN phone 49 0 89 726 3718 0 Version 2 0 Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle between Absorbance and T mode 4 Print graph greyed out if no data are available T Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in User Methods 1 9 press the down arrow and enter name 9 Auto print toggles auto print on off Exit options by pressing or wait Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 41 Functions 5 2 Concentration This makes simple concentration measurements on samples by
55. he 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 Auto print toggles auto print on off Exit options by pressing A or wait Peak Detection Shortcut button 4 Auto Detect Peaks Turns on and off the automatic peak detection The following options determine how peaks are detected Minimum Peak Height Minimum height the peak has to be above the higher of the two adjacent minima for the peak to be detected Minimum Peak Width Minimum width of the peak as determined by the difference in wavelength between the highest of the two adjacent minima and the opposing intersection of that higher minimum level and the peak profile See the screen displayed below Peak Detect on Zoom Determines whether peaks are re assessed and tabulated when the user zooms into a region of the wavescan lf 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 disp
56. he 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 I g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed Decimal Points DP to be selected from O to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98 768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters OR Cancel Q Press Next to enter the next screen Lowry Parameters Step 8 Select the Calibration mode either standards measure prepared standards manual keypad data entry or new standards using a saved method previous values are Regression blanked new standard can be measured eae Step 9 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 Replicates mm S Step 10 Press Next d to enter the Standards screen OR Press L Cancel Cancel to cancel selections and return to the Protein folder Standards Screen Standards Screen Lowry Standards Step 11 Enter the concentration values by using the keypad numbers and the up and down arro
57. if selected Step 13 If the absorbance value of the sample is not between 0 02 and 1 7 a Warning message and Instruction will be displayed in the top left corner of the result screen Please refer to 3 2 Software instructions for further information Step 14 Repeat for all samples samples Step 15 Press Options to display available Options which are described below Step 16 Press and confirm with d to return to the Protein folder EG phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 21 LabelGuard Cuvette Applications Step 17 Options select using key pad numbers Options select using key pad numbers 1 Return to parameters screen P 2 Print result via selected method pLi EE 3 Toggle graph on off The graph shows a wavescan plot across z z the scan range 210 nm to 760 nm with cursors denoting 260 nm i and 280 nm ER Print Data Gris 4 Toggle on off to display the data table only in the print out Default setting prints always the graph plus the data T Sample number add a prefix to the sample number and reset eh Sample Number the incrementing number to the desired value Ch Save Method 8 Save method use the left and right arrows to select a folder to Auto Print store in User Methods 1 9 press the down arrow and enter name sone e 9 Auto print toggles auto print on off Exit op
58. ight arrows Enter the second Wavelength as above Select whether a Background correction is applied to both wavelengths 1 and 2 using the left and right arrows lf background correction is On Enter the third Wavelength from which the background correction will be obtained Press Next di to enter the next screen OR Press Cancel to return to the Functions folder Select the Pathlength using the left and right arrows Options are 0 1 0 2 1 and 2 mm for LabelGuard applications and 5 or 10 mm for cuvette applications Dilution Factor known Enter a Dilution factor by using the keypad numbers within the range 1 00 9999 OR Calculate Dilution Factor Press the Options key Enter the Volume of the sample range 0 01 9999 using the keypad numbers Enter the volume of Diluent range 0 01 9999 by using the keypad numbers Press OK d to calculate the dilution factor and return to the Parameters screen OR press Cancel to cancel selections Select units of measurement using left and right arrows Options are ug ml ng ul ug l Enter the factor using the keypad numbers Range 0 001 to 9999 Press OK gt to enter the results screen OR Cancel to return to the Functions folder Email info implen de www implen de Functions Results Screen 0 258 A 0 1665 Absorbance R atio Concentration di OT gt 12 5 ug ml Options select using key pad numbers Parameters Print Graph Sam
59. ing the left and right arrows Step 2 Adjust the Contrast using the left and right arrows sled ltt Step 3 Press OK to store the settings and return to the Utilities folder Contrast INL CN phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 60 Utilities 7 6 About About ManoPhotometer M Displays the instrument serial number and software version Serial Number 16rrr215 gt Ir l S Press OK to close the window and return to the Utilities folder 3 Wersion P22 2 0 0 i f or walt Build 4 i www implen de di OK INL CN phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 61 Utilities 8 ACCESSORIES Certified Didymiumglasfilter for the control of wavelength and photometric accuracy for the NanoPhotometer NP HDF 100 NanoPhotometer Software Package NP SW100 Thermal Paper Printer module for the NanoPhotometer B 80 3003 84 Replacement Paper Rolls for Thermal Paper Printer pack of 5 VI G1D 04712 Bluetooth connection module B 80 3003 96 SD Memory Card Module B 80 3005 05 Dust cover for the NanoPhotometer B 80 3004 02 Lid 5 2 mm lid for LabelGuard Microliter Cell LG 1L00 L2 Lid 100 0 1 mm lid for LabelGuard Microliter Cell LG 100 LO1 9 MAINTENANCE 9 1 Maintenance free Technology The NanoPhotometer technology is maintenance free Regular maintenance or calibratio
60. ing to enter the Options menu EG phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 8 Introduction 3 THE LABELGUARD MICROLITER CELL With its innovative optical pathway the cell is designed for optimum measurement results with submicroliter sample volumes ranging from O 7 ul up to 10 ul of undiluted sample Due to a pathlength of 0 1 mm 0 2 mm 1mm and 2 mm the cell is offering an automatic dilution of 1 100 1 50 1 10 and 1 5 in comparison to a standard cuvette measurement Because the measurements are processed with undiluted samples the reproducibility of the results is extremely high If desired samples can be retrieved after the measurement for further processing The LabelGuard Microliter Cell can be used for all UV Vis analysis utilizing the wavelength range of 190 to 1100 nm The LabelGuard Microliter 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 and Lid 100 0 1 mm pathlength are optional The dilution factor lid factor is printed on the lid Please make sure that you use the appropriate lid for your sample Note The LabelGuard Microliter Cell is delivered with two adapters The centre height can be adjusted to 8 5 mm no adapter 15 mm Z15 or 20 mm Z20 Before starting make sure that the centre height of the LabelGuard is adjusted to the right setting For t
61. int 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 Auto print toggles auto print on off Exit options by pressing A OR wait Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 19 LabelGuard Cuvette Applications 4 2 Protein Determination 4 2 1 General Information Protein determination at 280 nm LabelGuard 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 absorption value for a particular protein must be determined The presence of nucleic acid in the protein solution can have a significant effect due to strong nucleotide absorbance at 280 nm This can be compensated by measuring Abs 260 and applying the equation of Christian and Warburg for the protein crystalline yeast enolase Biochemische Zeitung 310 384 1941 Protein mg ml 1 55 Abs 280 0 76 Abs 260 or Protein conc A280 Factor Abs 280 A260 Factor Abs 260 with A260 Factor Correction factor for absorbance reading at 260nm and A280 Factor Correction factor for absorbance reading at 280nm This equation can
62. irm with d 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 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 Auto print toggles auto print on off Exit options by pressing GA or wait Fax 49 0 89 726 3718 54 Page 51 Email info implen de www implen de Functions 5 6 Multiple Wavelength This makes up to 5 absorbance measurements on the same sample The procedure is as follows Parameter Screen Parameter Screen Multi wavelength Parameters Step 1 Press 3 to select Functions Step 2 Press 6 to select Multi Wavelength Step 3 Select the number of Wavelengths Step 4 Select the Pathlength using the left and right arrows et ee Options are 0 1 0 2 1 and 2 mm for LabelGuard applications and 5 or 10 mm for cuvette applications Step 5 Press OK to enter the next screen Step 6 Enter the first Wavelength using either the number keys or the left and right arrows Step 7 Enter the second Wavelength as above and repeat fo
63. known extinction factor of the oligo used factor range 0 01 to 9999 for ratio 1 extinction coefficient 10 Step 10 Press OK d to enter the Results screen OR Cancel B to return to the Nucleic Acids folder Cuvette Applications Oligo Parameters D E Factor Sai A Results Screen Results Screen Step 11 Insert the reference sample Press Blank Key This will be used for all subsequent samples until changed Step 12 Insert sample and press this measures at the selected wavelengths and displays the results The sample concentration and the ratio of A260 A280 and i A260 A230 are calculated corrected by the background wavelength value if selected Step 13 If the absorbance value of the sample is not between 0 02 and 1 7 a Warning message and Instruction will be displayed in the top left corner of the result screen Please refer to 3 2 Software instructions for further information Step 14 Repeat for all samples Step 15 Press Options to display available Options which are described below Step 16 Press and confirm with d to return to the Nucleic Acids folder EG phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 16 LabelGuard Cuvette Applications Options select using key pad numbers Farameters Frint Graph Frint Data Only Sample Number Save Method Auto Frint INL CN phone 49 0 89 726 37
64. laying the measured peak height and horizontal dashed lines showing the peak width Pressing Cancel ignores the selection pressing d accepts them Email info implen de www implen de Page 45 Functions Add Peak Shortcut button 5 a avescan User Defined Peak d HN Parameters Print Abs SI Peak Detection Delete Peak Graph Scale Sample Murober Save Method Auto F ririt 000000000 E D Ce Ca Wee WEE S A 495rm Abs a A Graph Scale Shortcut button 6 Graph Scale zoom Mode H axis limits y axis limits SSES FOO ppm INL CN phone 49 0 89 726 3718 O Version 2 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 x amp y axis expands the display around the cursor measurement point whilst the other options select the absorbance or wavelength axes respectively With x or y axis
65. 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 d accepts it and displays the required graph Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 46 Functions 5 4 Kinetics Simple kinetics studies where the change in absorbance needs to be followed as a function of time at a fixed wavelength can be readily performed Reagent test kits are routinely used for the enzymatic determination of compounds in food beverage and clinical laboratories by measuring NAD NADH conversion at 340 nm The change in absorbance over a specified time period can be used to provide useful information when an appropriate factor defined in the reagent kit protocol is applied Reaction rate and enzyme activity can be calculated if the factor used takes account of the absorbance difference per unit time aS Opposed to the absorbance difference per se For this reason the change in absorbance per minute AA min concentration AA min x factor and correlation coefficient calculated from a best fit of the data points are displayed They may not be relevant for simple kinetics experiments The procedure to define a new method is as follows Parameter Screen Kinetics Param
66. measuring the amount of light that has passed through a sample relative to a reference this can be air Concentration is obtained by multiplying the measured absorbance at a specific wavelength by a factor The factor may be known in advance or may be calculated by the instrument by measuring a standard of known concentration The procedure is as follows Parameter Screen Concentration Parameters Parameter Screen Step 1 Press 3 to select Functions DEE 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 pone left and right arrows Step 5 if Factor is selected Enter the Factor using the keypad numbers Range 0 001 to 9999 Use the C button to delete the last digit entered eme E Step 6 if Standard is selected Enter the concentration using keypad numbers Range 0 01 9999 Use the C button to delete the last digit entered Concentration 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 I g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed Decimal Points DP to be selected from
67. n LabelGuard Applications dsD0N4 Oyve Parameters Lid Factor Units Dilution Factor Factor Background Dyve Type Cuvette Applications dsD0N4 Ove Parameters Pathlength Units Dilution Factor Factor Background Dyve Trpe Results Screen dsDNA Dye aen area Sampe ER A250 0 046 A Concentration 0 0035 4 D 22 moll 0 132 4 0 066 A FO Cys ADyve 550 0 132 4 52 5 AVBOSATEO 7 587 5 Oye Concentration AzZ60 A 230 1 566 260 230 SEN MALEN phone 49 0 89 726 3718 O Version 2 0 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 Step 12 Step 13 Parameter Screen Press 1 for LabelGuard OR 2 for Cuvette folder Press 1 to select Nucleic Acids folder Press 5 6 7 or 8 to select one of the dye incorporation methods Using the LabelGuard Applications select the Lid Factor as described under 3 2 Using Cuvette Applications select Pathlength using the left and right arrows Options are 5 mm or 10 mm Select Dilution Factor Units and Factor as described under 4 1 2 4 1 3 Background correction at 320 nm is recommended to be switched on Select the appropriate Dye Type 10 different AlexaFluors 4 Cy Dyes 6 Oyster Dyes and Texas Red are programmed with their corresponding maximum absorbance wavelength dye dependent correction factor at 260 nm and dye dependent extinction coefficient For further details please refer to 11 2 Nucleic
68. n is not necessary For facilities that are working according to national as well as international guidelines and standards like 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 secondary standards as an optional accessory These NanoPhotometer Didymiumglassfilters 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 9 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 9 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 Changing cell holder or removal for cleaning This can be removed by undoing the appropriate screws on the bottom of the instrument MELEN phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 62 Accessories 10 SPECIFICATION AND WARRANTY Technical Specifications
69. nd 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 mol 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 d to store the chosen parameters OR Cancel Q Press Next to enter the next screen Select the Calibration mode either standards measure prepared standards manual keypad data entry or new standards 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 gt to enter the Standards screen OR Press Cancel to cancel selections and return to the Protein folder Standards Screen Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range 0 001 to 9999 C button backspac
70. nfo implen de www implen de Version 2 0 Page 58 Utilities 7 1 Date and Time The procedure is as follows Date and Time Minute 7 2 Regional 2006 GE Cancel Sets Number Format The procedure is as follows Regional Number Format 9933 9 E Dk D Cancel 7 3 Printer Sets up printing options The procedure is as follows Auto Print ni Printer Dr Dk D Cancel ces Version 2 0 phone 49 0 89 726 3718 O Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 1 Step 2 Step 1 Step 2 Step 3 Fax 49 0 89 726 3718 54 Page 59 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 di to store the settings and return to the Utilities folder OR Press Cancel to return to the Utilities folder without storing the time Set the Number Format decimal point style Options are d D d D Or Press OK di to store the settings and return to the Utilities folder OR Press Cancel Q to return to the Utilities folder without storing the settings Select 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
71. ngs If a solution is too dilute the readings will be at the instrument s detection limit and results may vary as there is less distinction of the 260 peak and the 280 slope from the background absorbance This is one reason why the Abs 260 value 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 Ce phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 12 LabelGuard Cuvette Applications at this wavelength When measuring RNA samples the 260 230 ratio should be gt 2 0 a ratio lower than this is generally indicative of contamination with guanidinium thiocyanate a reagent commonly used in RNA purification and which absorbs over the 230 260 nm range A wavelength scan of the nucleic acid is particularly useful for RNA samples e 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 e 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
72. o 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 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 Auto print toggles auto print on off Exit options by pressing Q or wait Fax 49 0 89 726 3718 54 Page 33 Email info implen de www implen de LabelGuard Cuvette Applications 4 2 7 Biuret Assay The colorimetric Biuret assay is not recommended with the LabelGuard Microliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen Biuret Parameters Parameter Screen Step 1 Press 2 to select Cuvette folder arenai Step 2 Press 2 to select Protein folder ae mi Step 3 Press 5 to select Biuret mode mm O Step 4 The default Wavelength setting is 546 nm Step 5 Enter the number of Standard concentration points 1 9 Ps to be used in the curve using the keypad numbers or left and right arrows Step 6 Select Pathlength using the left and right arrows Options are 5mm or 10 mm Step 7 Units
73. one 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 64 Appendix The following dye types and parameters are pre programmed in the NanoPhotometer Dye dependent Dye dependent correction aa Absorption maximum extinction coefficient factor 260 nm Dye Type Dye nm Edye CF bye Mexa Fluor647 eso 29000 0 00 Mexa Fuoree0 mu mmm oo Mexa Fuores0 mu meo o0 cys Oyster 500 503 78000 Oysters eo 2000 oa In all formulas the molar dye dependent extinction coefficient is used Ce phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 65 Appendix 11 3 Protein fluorescent dye incorporation To determine the protein concentration and the dye concentration after labelling a modification of the Lambert Beer equation is used e Calculation A280 factor A280 Factor molecular weight protein dye dependent extinction coefficient e Calculation of the Protein Concentration with correction of dye contribution to the 280 nm absorbance reading Cprot A280 CFaye Amax aye A280 Factor Lid factor Dilution factor Cprot protein concentration mg ml A280 absorbance AU of proteins CFaye dye dependent correction factor at 280 nm Amax dye absorbance at absorption maximum of the dye AU Lid factor virtual dilution factor 5 10 50 or 100 Dependent on the used LabelGuard Lid e Calculation of the Dye conc
74. or 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 LabelGuard Microliter Cell is not recommended for optical density measurements of cell culture solutions Ce phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 37 LabelGuard Cuvette Applications 4 3 2 Analysis of Bacterial Growth The procedure is as follows Parameter Screen Step 1 Press 2 to select Cuvette Applications Parameter Screen Step 2 Press 3 to select OD 600 OD 600 Parameters Step 3 Select the Wavelength Default value is 600 nm Range is 200 nm to 950 nm wavelength Step 4 Enter the Correction factor to compensate for different B00 nm optical configurations between this and other instruments Default value is 1 Correction Step 5 Select the Units Options are OD or cells ml If cells ml is 1 000 selected two further parameters are displayed Step 6 if cells ml selected Enter the Factor using the keypad numbers Range 0 00 to 9999 C button backspaces and OD 600 Parameters clears the last digit entered Step 7 if cells ml selected Select the Multiplier using the left Wavelength Factor and right arrows Options are 1000 or 1 000 000 600 run Factor and Multiplier define the conversion of the measured OD to the number of cells per millilitre eg 1 Correction Mul
75. ows ug ml ug ul pmol ul mg dl mmol l umol I g l mg l ug l U I pom 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 98 768 2 will display as 98768 even with 1 decimal point selected Press OK dn to store the chosen parameters OR Cancel GA 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 9999 Select the Pathlength using the left and right arrows Options are 0 1 0 2 1 and 2 mm for LabelGuard applications and 5 or 10 mm for cuvette applications Press Next d to enter the Results screen OR Press Cancel to return to the Parameters screen EG phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 47 Functions Results Screen Ag 4 dA 0 001 0 006 0 005 01957 Sample 1 Time 00 00 10 Abs 0 226 Options select using key pad numbers Parameters Print Print Data Set tO At Cursor Get tn At Cursor Slope Sample Mumber Save felethod Subo Frint 6090560006 KC Bee e le ee a Min n a n a n a n a a a a a a n a a a a n n n n n n n n n n n n a a
76. ple Huber Save Method Ubo Frint Ce Version 2 0 phone 49 0 89 726 3718 O Step 15 Step 16 Step 17 Step 18 Step 19 Results Screen Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Insert sample and press d 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 Press Options to display available Options which are described below Press and confirm with d 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 Auto print toggles auto print on off Exit options by pressing A or wait Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 55 Functions 6 USER METHODS These folders are the storage locations for any user modified Applications Methods that are saved in the Options menu
77. plen de Version 2 0 Page 9 LabelGuard Microliter Cell 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 LabelGuard Applications and Cuvette Applications are very similar concerning the analysis of dsDNA ssDNA RNA Oligonucleotides protein UV and protein dye analysis This section describes the specific features which have to be considered using the LabelGuard Microliter Cell For general information please follow the detailed instructions under LabelGuard Applications and Cuvette Applications The procedure is as follows Exemplary Parameter Screen Parameter Screen Step 1 Press 1 to select LabelGuard Applications folder Step 2 Press 1 to select Nucleic Acids folder OR 2 to select Protein folder Step 3 Select the method you want to use by pressing the corresponding number Dilution Factor Factor dsDNA Parameters Step 4 Select the Lid Factor using the left and right arrows Lid Lid Factor Dilution 100 optional 1 100 Background ES 5 optional Step 5 Select subsequent parameters and specifications as described under 4 LabelGuard Applications and Cuvette Applications After the selections are confirmed the results screen displays in top let corner the chosen Lid and the required sample volume EG phone 49 0 89 726 3718 O Fax 49
78. plications 5 FUNCTIONS Survey of the available Functions Functions e wavelength B sah Multi wavelength Fins Concentration F A bpsorbance Ratio 4a Absorbance or T transmission at a Absorbance or T transmission at a single user defined wavelength 00 user Absorbance or T transmission at a single user defined wavelength 00 wavelength ee eg 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 A Kinetic colorimetric assay either rate or end value based E 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 the History parameter to on see Preferences later will cause the instrument to store it s 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 MELE
79. r a DK e Cancel the number of wavelengths selected up to 5 Step 8 Press OK d to enter the results screen OR Press Cancel Multi Wavelength Parameters to return to the Functions folder Results Screen Step 9 Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Hulti wavelength Step 10 Insert sample and press Abs ao WS O Step 11 Repeat for all samples A scan plot covering the range of E age wavelengths selected with cursors at the relevant La wavelengths and a table of values is displayed P eae E Step 12 Press Options to display available Options which are ee described below H HDD s00 BD Step 13 Press and confirm with d to return to the Functions folder Query needs confirmation to avoid unintended escaping the application Multi wavelength Sample H ml HI iE yh Ri wl E 300 nm 0 018 400 nm 0 016 500 nm 0 013 600 nm 0 013 EG phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 52 Functions Options select using key pad numbers Farameters Frint Frint Graph Sample Mumber Saue Method Uto Frint INL CN phone 49 0 89 726 3718 0 Version 2 0 Options select using key pad numbers 1 Return to parameters screen
80. 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 d to accept the calibration and go to the Results screen see below OR Press Back to cancel selections and return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Press d 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 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 Press to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Email info implen de www implen de LabelGuard Cuvette Applications Calibration Screen manual entry Bradford Calibration 0 058 4 gt 0 156 4 HE i Ba 0 320 4 W 0 463 A Zi A I 0 7154 th Ap a CS DH DR D Lo Lg LN e Back a Ke E
81. 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 Ed Press OK d to store the settings and return to the Utilities folder OR Press Cancel to return to the Utilities folder without storing the settings Email info implen de www implen de Utilities ZA Preferences Sets user preferences The procedure is as follows Preferences Step 1 Select Games function This determines whether the games folder is displayed or not Options are yes or no Auto Standby Step 2 Define the Screen layout Theme of folders Options are either a grid format or a list Step 3 Select History whether to use previously entered Baseline Compensation parameters memory function or to return to default settings Step 4 Select whether to use a Standby mode after defined History periods Options are 1 hour 2 hours at night or off Step 5 Select Baseline Compensation to improve value stability and to overcome background effects Step 6 Press OK di to store the settings and return to the Utilities folder OR Press Cancel to return to the Utilities folder without storing the settings 7 5 Contrast Ambient temperature can affect the display This function can optimize the display for local conditions The procedure is as follows Contrast Step 1 Adjust the Brightness us
82. tails please refer to the NanoPhotometer 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 d to save the method OR B to return to the User Methods folder EG phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 56 User methods Delete Method 1 Select the method to be deleted using the key pad numbers 2 Select options and press 1 Delete Method Methods Methods 1 3 Press d to delete the method OR Q to return to User oi Single wavelength Methods folder lt gt Delete Method Lock Method eh Unlock Method EG phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 57 User methods 7 UTILITIES Survey of the available Utilities Utilities O g ln Select screen layout themes history and Baseline Compensation Adjust screen contrast amp brightness Serial number and software version MELEN phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email i
83. thods methods e 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 optional NanoPhotometer Software Package NP SW100 is necessary to connect the NanoPhotometer to a PC The software enables the user to print through the PC directly to the printer that is connected to it Data may be stored as Excel spreadsheet EMF graphics file a comma delimited csv data file a tab delimited txt data file or in native NanoPhotometer 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 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 LabelGuard Microliter Cell and standard 10 mm pathlength quartz glass
84. tions by pressing GA or wait CES phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 22 LabelGuard Cuvette Applications 4 2 3 Protein UV Dye Method The procedure is as follows Parameter Screen LabelGuard Applications Protein Oye Parameters Step 1 Step 2 Lid Factor A280 Factor Step 3 Step 4 Dilution Factor Units Background Protein Ext Coefficient Step 5 Cuvette Applications Protein Oye Parameters Pathlength AZEO Factor Step 6 i Dilution Factor Units Step 7 Step 8 Background Protein Ext Coefficient Step 9 Step 10 Protein Oye Oye Parameters Step 11 Oye T ype Dye Abs Max Dye Ext Coefficient Step 12 Oye Correction E Cancel Version 2 0 phone 49 0 89 726 3718 O Page 23 Fax 49 0 89 726 3718 54 Parameter Screen Press 1 for LabelGuard OR 2 for Cuvette folder Press 2 to select Protein folder Press 2 to select Protein dye mode Using LabelGuard Applications select the Lid Factor as described under 3 2 A minimum of 2 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 9999 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 9999
85. tiplier OD 600 5 x 108 cells ml ioe AAA Step 8 Press OK d to enter the Results screen OR Press Cancel to cancel selections and return to the Cuvette Units Applications folder lt gt Ok Results Screen Results Screen Step 9 Insert the reference sample and press Blank key This Kette eege will be used for all subsequent samples until changed 550 nm Step 10 Insert the sample and press The wavelength Absorbance cells ml absorbance and OD6OO value is displayed coos Step 11 Repeat for all samples Step 12 Press Options to display available Options which are described below Step 13 Press and confirm with d to return to the Cuvette Applications folder Query needs confirmation to avoid o 1000 000 unintended escaping the application Options select using key pad numbers Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 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 Sample Humber 9 Auto print toggles auto print on off Save Method Awuto Print Exit options by pressing A or wait Farameters Ent EG phone 49 0 89 726 3718 O Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 38 LabelGuard Cuvette Ap
86. 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 d to accept the calibration and go to the Results screen see below OR Press Back to cancel selections and return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Insert the reference sample Press Blank key This will be used for all subsequent samples until changed Press d 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 Email info implen de www implen de LabelGuard Cuvette Applications Calibration Screen manual entry Biuret Calibr
87. to display the Run Standard screen Step 14 Run the standard by pressing d OR Press Cancel to return to the measure screen Step 15 Insert the sample and press di 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 Q and confirm with d 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 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 Auto print toggles auto print on off Exit options by pressing GA or wait Email info implen de www implen de Page 43 Functions 5 3 Wavescan An absorption spectrum can be obtained from your instrument enabling simple identification of peak height and position The procedure is as follows Parameter Screen Parameter Screen Wavescan Parameters Step 1 Press 3 to select Functions Step 2 Press 3 to select Wavescan SE EMS Step
88. tration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range 0 001 to 9999 C button backspaces and clears the last digit entered Press Next d to enter the Calibration screen If there are duplicate or non monotonic increasing entries the unit will beep and highlight the incorrect entry OR Press Back to return to the Parameter screen Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 28 LabelGuard Cuvette Applications Calibration Screen replicates off Bradford Calibration d DH kE WR LO le Lu LP Bradford Calibration 0 200 0 058 A A 0 400 0 1955 4 a DP K 0 600 0 330 A j 0 800 0 462 4 a HN Va 1 000 0 532 A y 1 400 O 7194 e D A 1 600 0 842 A Ta a2 HH O86 DR LO L LH LE De OK EA Back Ced D D DH O68 DR LO Le L Version 2 0 phone 49 0 89 726 3718 O Step 13 Step 14 Step 15 Step 16 Step 17 Step 18 Step 19 Step 20 Step 21 Fax 49 0 89 726 3718 54 Page 29 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
89. using the keypad numbers Range 1 00 to 9999 Use the C button to backspace and e cance 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 9999 Enter Cuvette Applications the volume of the diluent using the keypad numbers EE Range 0 01 to 9999 Press di to calculate the dilution factor and return to the Parameters screen OR Press Pathi th AZ60 Fact Cancel to cancel the selections and return to the Parameters screen emm Step 6 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 Step 7 Enter A260 Factor using the keypad numbers Default value is 0 76 Range is 1 00 to 9999 Step 8 Enter A280 Factor using the keypad numbers Default value is 1 55 range is 1 00 to 9999 Step 9 Select the Units of measurement using the left and right arrows Options mg ml ug ml ng ul and yg ul Step 10 Press OK to enter the Results screen OR Cancel to return to the Protein folder Results Screen Results Screen Step 11 Insert the reference sample Press Blank Key This will be used for all subsequent samples until changed Protein Uv ae Step 12 Insert sample and press d This measures at both 260 and 280 nm wavelengths and displays the result Protein concentration is calculated corrected by background wavelength value
90. w implen de LabelGuard Cuvette Applications Results Screen 3 5 Protein Oye 4760 163 4 Protein Concentration 4D0ye 654 0 1764 A 1 12 mami T 36 pmol pl Degree of Labelling Options select using key pad numbers Farameters Print Graph Print Data Only Sample Mumber Save Method S ubo Frint a Vd E G O E E INL CN phone 49 0 89 726 3718 0 Version 2 0 Results Screen Step 13 Insert the reference sample Press Blank Key This will be used for all subsequent samples until changed Step 14 Insert sample and press d This measures at 260nm 280nm 320nm and the dye specific wavelength and displays the result Protein concentration corrected by background wavelength value if selected dye concentration and degree of labelling is calculated Step 15 If the absorbance value of the sample is not between 0 02 and 1 7 a Warning message and Instruction will be displayed in the top left corner of the result screen Please refer to 3 2 Software instructions for further information Step 16 Repeat for all samples Step 17 Press Options to display available Options which are described below Step 18 Press and confirm with d to return to the Protein folder Options select using key pad numbers 1 Return to parameters screen 2 Print result via selected method 3 Toggle graph on off The graph shows a wavescan plot across the scan range 210 nm to 760 nm
91. with cursors denoting 260 nm and 280 nm 4 Toggle on off to display the data table only in the print out Default setting prints always the graph plus the data 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 Auto print toggles auto print on off Exit options by pressing GA or wait Fax 49 0 89 726 3718 54 Email info implen de www implen de Page 24 LabelGuard Cuvette Applications 4 2 4 BCA Assay The colorimetric BCA assay is not recommended with the LabelGuard Microliter Cell Please use Cuvette Applications The procedure is as follows Parameter Screen BCA Parameters Standards Calibration 4 Standards Replicates Standards Screen EEN Version 2 0 BCA Standards phone 49 0 89 726 3718 O Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 Step 12 Step 13 Std 3 Std 6 0 600 1 400 Ee Parameter Screen Press 2 to select Cuvette folder Press 2 to select Protein folder Press 2 to select BCA mode The default Wavelength setting is 562 nm Enter the number of Standard concentration points 1 9 to be used in the curve using the keypad numbers or left and right arrows Select Pathlength using the left a
92. ws to move between SE the different standard boxes Range 0 001 to 9999 C button backspaces and clears the last digit entered Std 2 Std 5 Step 12 Press Next d 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 Std 3 Std 6 Back to return to the Parameter screen Ce phone 49 0 89 726 3718 0 Fax 49 0 89 726 3718 54 Email info implen de www implen de Version 2 0 Page 31 LabelGuard Cuvette Applications Calibration Screen replicates off Lowry Calibration d DH 0 6 DR LH Le LH Lowry Calibration 0 20 Zu 0 200 O01 A S 0 400 0 056 A RH 0 15 0 600 0 095 4 A 0 800 0 1324 ra HI i 1 000 0 169 4 n 1 400 0 205 A Zei 0 05 Ba a a2 DH 0 6 0 8 1 0 Lg LN ok Calibration Screen replicates on a2 HH 0 6 0 8 1 0 Lg Lu e Back Step 21 Version 2 0 Page 32 phone 49 0 89 726 3718 O Step 13 Step 14 Step 15 Step 16 Step 17 Step 18 Step 19 Step 20 Fax 49 0 89 726 3718 54 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 grap

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