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NanoVue - GE Healthcare Life Sciences

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1. Replace the top cover plate invert the instrument and replace the cap head screws at locations A and B shown in step 1 8 2 Bluetooth Accessory Installation Step 1 As for section 8 1 remove the power cable and turn the instrument over onto a soft surface taking care not to damage the sampling head Release the outer most screws using the Allen key provided Turn the instrument back over and remove the accessory covers Step 2 Turn the instrument back over and lift the accessory cover vertically upwards to remove Remove the tie wrap from the cable Step 3 Plug the accessory cable into the Bluetooth module Step 4 Note the slots in the base of the case The two lugs tee _ on the Bluetooth module plug into these HVA a a E Waitt i myn _ a a rr pa ae dog Figure 19 Step 5 Load the rear cover into the two slots provided note the large flange is to face upwards i Figure 20 Version 4 0 Page 77 Version 4 0 Figure 21 Page 78 Step 6 Re fit the top plate Step 7 Invert the instrument carefully and replace the cap head screws Step 8 Switch the instrument on and go to the preferences page under utilities instrument and select the Bluetooth option Printer Auto Print Printer 4 Computer Bluetooth F 8 3 After Sales Support For all technical support and advice on NanoVue please contact your supplier GE H
2. This could be of the following form 3 5 Lamp replacement The xenon lamp should not need replacement until used for several years In the unlikely event that it does fail this should be undertaken by a service engineer from your supplier 3 6 Replacing the printer paper Spare paper for the printer 20 rolls is available from your supplier under part number 28918226 Step 1 Keep the power on Lift off the paper cover Step 2 Lock the platen horizontal position and feed the paper into the slot The drive will engage automatically and take up the paper Step 3 It may help to release the platen lock turn flat green catch clockwise and turn the green knob manually Step 4 Replace the cover Version 4 0 Page 15 4 LIFE SCIENCE 4 1 Filesystem The Life Science Screen is organized into six sub folders as shown below ok eff eff 0NA RNA Oligo olki ef ef Tm Calculation Cy Ove Protein Folder Keypad a Application DNA Concentration and purity check for DNA samples RNA Concentration and purity check for RNA samples Oligo Concentration and purity check for Oligonucleotide samples Tm Calculation DNA melting point calculator Cy Dye Labeling efficiency measurement Protein Folder containing methods for protein determination Pressing 6 enters the Protein sub folder which contains various standard methods for protein determination Life Science Protein ols ef elz BCA Protein Ly Protein 4280 o
3. Press Cancel to return to the Utilities Screen without storing the settings The procedure is as follows 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 tt is off printing has to be initiated manually This can also be set using the Options key KEKA in each application or method The default is Off Press the down arrow Select how the data are sent Options are Built in internal printer or to a computer via USB port or Bluetooth Press OK to store the settings and return to the Utilities Screen OR Press Cancel to return to the Utilities Screen without storing the settings Page 69 7 4 Preferences Sets user Preferences move around and 1 Games this determines whether the games folder is toggle on and off with the arrows displayed or not Options are yes or no 2 Theme defines the screen layout of folders Options are either a grid format default or a list Preferences a History selects whether to use previously entered parameters when the instrument is switched on or whether to use defaults 4 Auto Standby selects whether to use a standby mode oh after defined periods Options are 1 hour 2 hours at night or Off History Quality Assurance 5 Lid Switch selects whether Auto Read is on or off When on readings will automatically be taken when the sampling head is lowered
4. 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 0 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 Standard Curve Parameters decimal point selected Press OK to store the chosen parameters or Cancel E Step 4 Enter the Pathlength Options are 0 5mm or 0 2mm Press Next to move on to the next Parameters Screen or Ese Cancel to return to the Applications Screen Step 5 Select the type of curve fit using the left and right arrows Options Regression straight line Zero Regression this forces the straight line through the origin Interpolated or Cubic Spline Step 6 i tas A Select the calibration mode Either Standards measure prepared standards Manual keypad data entry or New Standards means new standards are measured each time the method is used Press the down arrow Curve Fit 4 Zero Regression fF Calibration Standards Step 7 if Standards has been selected in step 5 Select the number of standards to be measured and averaged at each standard concentration point Can be Off 2 or 3 Replicates Press Next to enter the Standards Screen OR Press Cancel to cancel selections and return to the Applications Screen Version 4 0 Page 60 Standard Curv
5. 5 2 Single Wavelength This makes 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 Note that the values reported are NOT nomalized to a 10mm pathlength so if comparison is being made to other systems the appropriate factor should be used to convert them x20 for 0 5mm pathlength x50 for 0 2mm The procedure is as follows Single wavelength Parameters wavelength Mode 4bsorbance Pathlength 0 5 mm Version 4 0 Single wavelength Single wavelength Step 1 Set Wavelength by using keypad numbers or left and right arrows range 190 1100nm Press the down arrow key Step 2 select the mode Absorbance or T using the left and right arrows Step 3 select Pathlength from 0 5mm or 0 2mm Step 4 To enter the Results Screen with the selected parameters press OK OR Cancel the selections and return to the Applications Screen by pressing Cancel E Step 5 Pipette on the reference sample and lower the sampling head lf Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 6 Clean the top and bottom plates pipette on the sample and lower the sampling head If Auto Read is off press FE Repeat step 6 for all samples Results Scre
6. If this happens see troubleshooting Section 9 to adjust the pathlengths If the instrument has a Printer fitted a hard copy of the calibration results can be obtained automatically upon exiting the lt application Ese if Auto Print is selected or by pressing Options Kk Print 2 If the instrument has no Printer fitted the same output can be gathered by the PVC application running on a neighboring PC Figure 11 Pathlength Calibration Product HanoVue Version 4 62 V1 7 serial Number 54321 Date 23 August 4007 Time 10 21 24 Instrument Calibration Pass 23 August 4007 10 10 38 Certificate ID PHD Calibration value 0 978 10 5 mm 10 2 mm Status H to 4ay At0 199 AIDE iz 0 495 A 0 194 AIOK 3 10 497 A 10 194 AIOK i4 10 495 A 10 192 AIOK i5 0 490 A 0 193 AIOK i6 10 488 A 0 195 AIOK i7 10 492 A 10 199 AIOK ia 0 481 AO 195 AOK 9 10 491 A 0 196 ADK 110 0 494 A 10 194 AIOK Mean 0 491 AiO 195 AL SD f0 9 11 1 iPathio 502 0 199 Date S 23 August Z00T7 Time 10 12 20 iCalibration Pass i Version 4 0 Page 72 7 8 Games 1 Spectro Blocks om eff Spectro Blocks Sudoku Score 480 1 Hew Game 2 Options 3 Exit Classic block dropping game Follow the instructions Press Cancel to return to the Utilities Screen without storing the settings Version 4 0 Page 73 2 Sudoku Can be set up as Computer Mode 50 preset games or User Mode enter your own patter
7. 33 the range is 0 01 to 9999 OR Oligo Parameters Step 6 Units pmol ul Enter the proportions of bases present using the keypad gle bid hh ue numbers and up and down arrows to move between boxes The default is 10 for each the range is 0 to 9999 Step 7 Dilution Factor 10 Press OK to enter the Results Screen and start making measurements Background OR Ese io return to the Life Science Screen Version 4 0 Page 23 OO tolon Version 4 0 Parameters F rint Graph Sample Mumber Save Method Subo Frint Results Screen Step 8 Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 9 Clean the top and bottom plates pipette on the sample and lower the sampling head If Auto Read is off press This measures at the selected wavelengths and displays the results The ratio of the absorbance values at wavelengths 1 and 2 are calculated The Concentration is based on the absorbance at wavelength 1 Repeat step 9 for all samples Press to return to the Life Science Screen Press II to display available Options which are described below Options select using keypad numbers 1 Return to Parameters Screen step 1 above 2 Print result via selected method 3 Toggle Graph
8. Method Aubo Prirt a O G O O 8 O Graph Scale X axis limits a ne amp Cancel ok Version 4 0 A458nm A 0 083 4 A458nm A 0 083 4 Add Peak shortcut button 5 Adds a used defined peak at the current cursor position The entry is then displayed in inverse coloring 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 Note Storing a method at this stage will save these user defined wavelengths each time the method is run the absorbance value at these wavelengths is reported 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 axes expands the display around the cursor measurement point whilst the other options select the absorbance or wavelength axes respectively With x or y axis limits set to on Zooming out will only be permitted to the set limits x y axis limits Setting x or y axis limits to On activates the start and finish points of the desired graph to user defined specific wavelengths and or absorbance values Pressing Cancel ignores the selection pressing OK
9. Std 3 Std 6 and clears the last digit entered Step 10 Standards Manual selected Press Next to enter the Calibration Screen If there are duplicate or non monotonic increasing entries the unit will beep and highlight the incorrect entry OR Press Back to return to the Parameters Screen Version 4 0 Page 37 BCA Calibration M Ara LH LE OE LO Le L4H BCA Calibration Standards 0 200 0 400 0 177 A tp 0 600 0 268 4 o 4 0 800 1 000 1 400 O 626 4 ps Fa 0 050 4 0 404 A O516 4 a HE LH LE OE LO Le LH mm o Li BCA Calibration 2 5 Replicates OO M S n fia LH LE QE LO Le LH BECA Calibration Standards 0 200 0 400 0 177 4 5 0 6 0 050 4 0 600 0288A ay 0 300 0 404 A 1 400 0 6264 WE j a Dd HE DLH LE OE LO Le LH 1 000 0 516 mm o Li Version 4 0 Calibration Screen Replicates off This shows the calibration values and allows standards to be measured or entered using the keypad numbers if calibration mode is Manual Step 11 Standards selected Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 12 standards selected Clean the top and bottom plates pipette on the standard and lower the s
10. TAC GAC Step 11 Press OK to select these parameters and start to measure Tm OR Press Cancel to return to the Parameters Screen Version 4 0 Page 27 Tm Calculation 2 35 4 Theoretical Abs Measured Tm Calculated Mv 2505 5 Calculated Factor Calculated Tm Parameters F rirt Go Sample Humber Save Method Subo Frimt n DoS Version 4 0 Results Screen Step 12 Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 13 Clean the top and bottom plates pipette on the sample and lower the sampling head If Auto Read is off press FWL The unit measures the absorbance and uses this information to calculate the Measured Tm Repeat step 13 for all samples Press to return to the Life Science Screen Press KHM to display available Options which are described below Options select using keypad numbers 1 Return to Parameters Screen step 1 above 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 alpha numeric keys to enter a name for the method and press Save 9 Auto Print toggles Auto Print on off Exit options by pressing or wai
11. accepts them and displays the required graph Page 56 5 5 Kinetics 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 Note that the system does not nomalize the result to a 10mm pathlength so the factor may need to be modified accordingly if brought over from another system The procedure to define a new method is as follows Kinetics Parameters 1 Kinetics Parameter 1 Screen Step 1 Wavelength Enter the Wavelength using the keypad numbers or the left and right arrows range 190 1100nm Press the down arrow Step 2 Delay Time Enter the Delay Time in seconds before measurements are an taken
12. are using the correct power supply It should be an 18V unit Make sure the sample plates are clean have been fitted securely and are well seated Is the xenon source firing when you press 7 Can you hear it buzz at start up Is the power supply securely connected to the instrument Turn off the instrument disconnect and reconnect the power supply and then turn on the instrument If the problem persists please contact your supplier Check that you are using the correct power supply It should be an 18V unit Make sure the sample plates are clean have been fitted securely and are well seated Is the xenon source firing when you press TT Can you hear it buzz at start up Is the power supply securely connected to the instrument Turn off the instrument disconnect and reconnect the power supply and then turn on the instrument lf the problem persists please contact your supplier Check that you are using the correct power supply It should be an 18V unit Make sure the sample plates are clean have been fitted securely and are well seated Is the xenon source firing when you press 1 Can you hear it buzz at start up Is the power supply securely connected to the instrument Turn off the instrument disconnect and reconnect the power supply and then turn on the instrument If the problem persists please contact your supplier Check that you are using the correct power supply It should be an 18V unit Make sure the sam
13. base of the sample plate and with the other hand gently lever down the tab at the top of the plate Replace the top sample plate Insert the bottom tab and hold this in place with one thumb and then gently push the tab at the top until the plate clicks into place Once in position two silver pins will be visible either side of the top sensor Remove the bottom sample plate Gently lever the plate upwards using the thumb tab at the front of the plate and then pull it towards you to remove it completely Replace the bottom sample plate Gently insert the tabs at the back of the plate as far as they will go and then push down the front of the sample plate so the tabs underneath are fully pushed in Always calibrate the system after replacing the sample plates Figure 8a Figure 8b Version 4 0 Page 14 3 3 Cleaning and general maintenance Before cleaning the case of the instrument switch off the instrument and disconnect the power cord Clean all external surfaces using a soft damp cloth A mild liquid detergent may be used to remove stubborn marks 3 4 Return for repair The responsibility for decontamination of the instrument lies with the customer The case may be cleaned with mild detergent or an alcohol such as ethanol or Isopropanol Other organic solvents are unsuitable Examination or repair of returned instruments cannot be undertaken unless they are accompanied by a decontamination certificate signed by a responsible person
14. be measured or entered using the keypad numbers if calibration mode is manual Step 11 Standards selected Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 12 Standards selected Clean the top and bottom plates pipette on the standard and lower the sampling head If Auto Read is off press MWL use C to clear previously stored results before measuring to measure the standard and store the result Repeat step 12 for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 Standards Manual selected When all standards are measured the OK box appears Press OK 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 Step 11 Standards selected Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the
15. be overwritten by moving the highlight bar pressing C to Figure 23 clear the line and re reading the calibration fluid Note that if you get a large change from reading to reading replace the calibration fluid Very bad readings gt 0 15A background absorbance or very low or high readings will be rejected by the instrument Step 6 When the adjustment is complete check by re referencing the system on distilled water and re measuring the calibration fluid checking the new values are within the specified limits If not repeat steps 4 and 5 Step 7 When complete re calibrate the unit by following the directions in section 7 7 Version 4 0 Page 80 9 2 Error Messages NanoVue has been designed for long life and high reliability In the event of a failure repair should be carried out by a trained service engineer from your supplier There are no components which can be replaced by the user except for accessories The following list of error messages has been provided for your information and to assist you when first contacting the technical support department of your supplier Cell holder obstructed no light Cell holder obstructed low light Cell holder obstructed signal fail Calibration problem UV on reference channel Calibration problem IR on reference channel Version 4 0 Check that you are using the correct power supply It should be an 18V unit Make sure the sample plates are clean have been fitted secure
16. ensure accurate results are obtained The small sample size required with NanoVue may allow modification of the manufacturer s protocols and considerable economies may be made at the user s discretion To use a zero concentration standard include it in the number of standards to be entered and enter 0 00 for concentration use this when required to enter standard 1 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 Use of Background Correction Background Correction at a wavelength well apart from the protein peak is used to compensate for the effects of background absorbance The procedure can adjust for the effects of turbidity stray particulates and high absorbance buffer solutions NanoVue uses Background Correction at 320nm It is particularly recommended since very small samples are particularly susceptible to stray particulates The Background function toggles On and Off with either left right arrows from the relevant page If it is used there will be different results from those when unused because Abs320 is subtracted from the Abs280 value prior to use in the above equations Background correction is not used in methods that use test kits such as the Bradford Biruet BCA and Lowry applications Version 4 0 Page 32 4 3 2 Protein UV
17. numbers or left and right arrows Press the down arrow Step 4 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 Kk and then use the left right arrows to select from ug ml ug l 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 0 to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters or Cancel E Step 5 Enter the Pathlength Options are 0 5mm or 0 2mm Press Next gt to move on to the next Parameters Screen or Ese Cancel to return to the Protein Screen Step 6 Enter the type of curve fit Options are Regression straight line Zero Regression forces the straight line through the origin Interpolated or Cubic Spline Press the down arrow Lowry Parameters Step T Select the Calibration mode either Standards measure Curve Fit prepared standards Manual keypad data entry go to step 9 or Regression New Standards means new standards are measured each time the method is used Calibration Step 8 Standards selected ese Select the number of Replicates using the left and right arrows This determin
18. on off The graph shows a Wavescan plot across the range 220nm to 320nm with cursors denoting 230 260 280 and if Background Correction is selected 320nm 7 Sample Number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save Method use the alpha numeric keys to enter a name for the method and press Save 9 Auto Print toggles Auto Print on off Exit options by pressing or wait Page 24 4 2 5 Tm Calculation This utility calculates the theoretical melting point from the base sequence of a primer It is done using nearest neighbor thermodynamic data for each base in the nucleotide chain in relation to its neighbor Breslauer et al Proc Natl Acad Sci USA Vol 83 p3746 1986 The data obtained are useful both in the characterization of oligonucleotides and in calculating T for primers used in PCR experiments The ACGT U sequence entered into the utility is used to calculate the theoretical Tm the theoretical absorbance Absorption units umol and the conversion factor ug ml This is because the stability of a bent and twisted sequence of bases such as an oligonucleotide is dependent on the actual base sequence The calculated thermodynamic interactions between adjacent base pairs have been shown to correlate well with experimental observations This utility uses matrices of Known published thermodynamic values and extinction coefficients to calculate Tm and the theo
19. on the sample and lower the sampling head If Auto Read is off press FEE The concentration of the sample is taken and displayed Repeat step 15 for all samples Press to return to the Protein Screen Press II to display available Options which are described below Options select using keypad numbers 1 Return to Parameters Screen step 1 above 2 Print result via selected method 3 Toggle Graph on off Displays the calibration graph cursors EP Parameters give values for last measured sample E Frin 7 Sample Number add a prefix to the sample number and E Graph reset the incrementing number to the desired value 8 Save Method use the alpha numeric keys to enter a name for the method and press Save 9 Auto Print toggles Auto Print on off Sample Mumber E Save Method auto Prin Exit Options by pressing E or wait Version 4 0 Page 39 4 3 5 Bradford The procedure is as follows Step 1 Bradford Parameters Press 4 to select Bradford method Step 2 Pathlenath The Wavelength for this method is fixed at 595nm Step 3 Enter the number of Standards 1 9 to be used in the curve using the keypad numbers or left and right arrows Press the down arrow Step 4 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 Kk and then use the left right arrows to select from ug ml yg ul pmol ul mg dl mmol l
20. the alphanumeric entry and arrow keys on the hard wearing spill proof membrane keypad Version 4 0 Page 9 oQ Life Science Display screen o Methods Arrow keys View options Key On Off key Arrow keys View options Bd BdM Alphanumeric keys Escape Cancel E Set Reference a Confirm selection Take measurement os Version 4 0 Alphanumeric keys Confirm selection NanoVue eG Applications Escape Cancel Utilities On Off Set reference Take measurement Action Turns the instrument on off Use the four arrow keys to navigate around the display and select the required setting from the active highlighted option View options for that application mode Some of these are common to all applications and are described below Options unique to an application are described in the relevant section Use these to enter parameters and to write text descriptions Use repeated key presses to cycle through lower case number and upper case Leave for 1 second before entering the next character Use the C key to backspace and the 1 key 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 at the current wavelength in the mode selected When in scan mode make a reference scan Confirms a selection Makes a measurement Page 10 Options Sub Menu select using keypad numbers QP Parame
21. 1 9 press the down arrow and enter name Auto Print toggles Auto Print on off Ese or wait Page 59 5 6 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 NanoVue has the advantage of being able to store this curve as a method using up to 9 standards If History is switched on see Preferences the unit will retain the previously used curve values 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 If you have History switched on and then select New Standards the old values will be cleared allowing the user to load a new curve Note that the Standard Curve function does not have background correction The procedure is as follows Step 1 Select the Wavelength using the keypad numbers or left and right arrows range 190 1100nm wavelength Pathlength Press the down arrow Step 2 Enter the number of Standards 1 9 to be used in the curve Press the down arrow Standard Curve Parameters Standards Step 3 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 Kk and then use the left right arrows to select from ug ml yg ul pmol ul mg dl mmol l umol g l mg l ug l U I ppm ppb
22. 6 0E 140 Le L4 Screen see below OR Press Back to return to the Standards Screen Results Screen Step 14 Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 15 Clean the top and bottom plates pipette on the sample and lower the sampling head If Auto Read is off press FE The concentration of the sample is taken and displayed Repeat step 15 for all samples Press to return to the Protein Screen Press KRM to display available Options which are described below Options select using keypad numbers 1 Parameters E Fin 1 Return to Parameters Screen step 1 above Graph 2 Print result via selected method 3 Toggle Graph on off Displays the calibration graph cursors give values for last measured sample 7 Sample Number add a prefix to the sample number and SAM Sample Number reset the incrementing number to the desired value E Sse Method 8 Save Method use the alpha numeric keys to enter a name EP sute Frint for the method and press Save 9 Auto Print toggles Auto Print on off Eso Exit Options by pressing or wait Version 4 0 Page 45 4 3 7 Biuret The procedure is as follows Biuret Parameters Biuret Parameters Curve Fit Regression Ca
23. A260 1 239 nal ul Version 4 0 Page 30 4 3 Proteins 4 3 1 Theory Protein Determination at 280nm Protein can be determined in the near UV at 280nm due to absorption by tyrosine tryptophan and phenylalanine amino acids The Abs280 varies greatly for different proteins due to their amino acid content and consequently the specific absorption value for a particular protein must be determined e The presence of nucleic acid in the protein solution can have a significant effect due to strong nucleotide absorbance at 280nm This can be compensated by measuring Abs260 and applying the equation of Christian and Warburg for the protein crystalline yeast enolase Biochemische Zeitung 310 384 1941 Protein mg ml 1 55 Abs280 0 76 Abs260 where means multiplied by or Protein conc Factor 1 Abs280 Factor 2 Abs260 e This equation can be applied to other proteins if the corresponding factors are known NanoVue can determine protein concentration at 280nm and uses the above equation as default The factors can be changed and the use of Background Correction at 320nm is optional e To customise the equation for a particular protein the absorbance values at 260 and 280nm should be determined at known protein concentrations to generate simple simultaneous equations solving these provides the two coefficients In cases where Factor 2 is found to be negative it should be set to zero since it means there is no contribu
24. ENI Select units of measurement using left and right arrows Options are ug ml ng ul ug l Press the down arrow Cancel Step 9 Enter the Factor using the keypad numbers range 0 001 to 9999 Press OK to enter the Results Screen or Cancel to return to the Applications Screen Version 4 0 Page 65 Results Screen Step 10 Pipette on the reference sample and lower the sampling head If Auto Read is off press the OA 100 T key This will be used for all subsequent samples until changed If QA is switched on the AssofAcen sample will need to be replaced and the 0A 100 T key pressed again Step 11 Clean the top and bottom plates pipette on the sample and Absorbance A atio lower the sampling head If Auto Read is off press l Concentration 2 380 pas mil Repeat Step 11 for all samples The absorbance at 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 to return to the Applications Screen Press KHK to display available Options which are described below Options select using keypad numbers 1 Return to Parameters Screen step 1 above 2 Print result via selected method z A 3 Toggle Graph on off Graph shows a wavescan plot across E Grah the selected wavelengths in place of the individual wavelength 7 Sample Number add a prefix to the sample number and reset the in
25. FICATION AND WARRANTY LEGAL Version 4 0 Page 3 67 INSTALLATION 1 1 Unpacking and positioning Remove the instrument from its packaging and inspect it for signs of damage If any are discovered inform your supplier immediately The instrument must be placed on a stable level surface that can take its weight 4 5 kg and positioned such that air can circulate freely around the casing Ensure your proposed installation site conforms to the environmental conditions for safe operation e The instrument is designed for indoor use only temperature range 5 C to 35 C and should be kept away from strong draughts e f 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 e A temperature change of no more than 4 C hour and a maximum relative humidity of 80 at 31 C decreasing linearly to 50 at 40 C are required If the instrument has just been unpacked or has been stored in a cold environment it should be allowed to come to thermal equilibrium for 2 3 hours in the laboratory before switching on This will prevent calibration failure as a result of internal condensation The instrument must be connected to the power supply with the power adaptor supplied The adaptor can be used on 90 240V 50 60HZz supplies It will become warm once plugged into the power suppl
26. NanoVue USER MANUAL TABLE OF CONTENTS 1 INSTALLATION 1 1 1 2 1 3 Unpacking and positioning Safety Declaration of conformity 2 INTRODUCTION Your NanoVue File system Data export Sample treatment Pathlength and absorbance normalization Auto Read Quality Assurance Keypad and display Software style 3 OPERATION AND MAINTENANCE Sample application guide Sample Plate replacement Cleaning and general maintenance Return for repair Lamp replacement Replacing the printer paper 4 LIFE SCIENCE 4 1 4 2 4 3 File system Nucleic acids 4 2 1 Theory 4 2 2 DNA measurement 4 2 3 RNA measurement 4 2 4 Oligonucleotide measurement 4 2 5 Tm Calculation 4 2 6 Cy Dye measurement Proteins 4 3 1 Theory 4 3 2 Protein UV 4 3 3 Protein A280 4 3 4 BCA 4 3 5 Bradford 4 3 6 Lowry 4 3 7 Biuret 5 APPLICATIONS Version 4 0 File system Single Wavelength Concentration Wavescan Kinetics Standard Curve Multiple Wavelength Absorbance Ratio Page 2 A A A O O 0 0O NN l 6 FAVOURITES AND METHODS 7 UTILITIES 7 1 Date and time 7 2 Regional 7 3 Printer 7 4 Preferences 7 5 Contrast 7 6 About 7 7 Pathlength calibration 7 8 Games 8 ACCESSORIES 8 1 Printer installation 8 2 Bluetooth accessory installation 8 3 After sales support 9 TROUBLESHOOTING 9 1 Pathlength calibration over range 9 2 Error messages 9 3 Fault analysis 9 4 Frequently asked questions 10 SPECI
27. This can be a maximum of 600 seconds 10 minutes Pers Press the down arrow Step 3 Duration Enter the time in minutes over which measurements are taken The software allows a maximum of 60 minutes however users should be aware that evaporation of the very small sample could have a significant effect on the results over such a long time period Press the down arrow Step 4 Interval Enter the Interval time in seconds between measurements using the left and right arrows Options are 1 5 10 20 30 or 60 seconds Press the down arrow Step 5 Press Next to go to the next Parameters Screen OR Press Cancel to return to the Applications Screen wavelength Delay Time Seconds Duration 1 Minute amp Cancel ei pciice Paramore 2 Kinetics Parameters 2 Screen Step 6 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 Mode Pathlength Version 4 0 Page 57 Kinetics Parameters 7 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 Kk and then use the left right arrows to select from ug ml ug l pmol ul mg dl mmol l umol g l mg l ug l U I ppm ppb
28. This is the Christian and Warburg assay discussed above The procedure is as follows Step 1 Protein UV Parameters Press 1 to select Protein UV mode Step 2 Select Pathlength using the left and right arrows Options are 0 5mm 0 2mm or Automatic Press the down arrow l Enter the Dilution Factor using the keypad numbers range 1 00 Te TT to 9999 Use the C button to backspace and clear the last digit OR Step 3 calculate Dilution Factor OF B Cancel p Press KHK to enter the Dilution Factor Screen shown to the left Enter the Volume of the sample using the keypad numbers range 0 01 to 9999 Protein UW Parameters Press the down arrow Enter the volume of the Diluent using the keypad numbers S range 0 01 to 9999 olume Press OK to calculate the Dilution Factor and return to the l Parameters Screen TRR OR Press to cancel the selections and return to the Parameters Screen Step 4 Select whether the Background Correction is to be used or not cance with the left and right arrows Press the down arrow Step 5 Enter the Abs260 Factor using the keypad numbers see method described in introduction The default value is 0 76 the range is 0 000 to 9999 Press the down arrow Step 6 Enter the Abs280 Factor using the keypad numbers see method described in introduction The default value is 1 55 the range is 1 000 to 9999 Press the down arrow Step 7 Select the Units of meas
29. a folder to store in Favourites Methods 1 9 press the down arrow and enter name 9 Auto Print toggles Auto Print on off pn Exit Options by pressing 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 Pk height Minimum height the peak has to be above the higher of the two adjacent minima for the peak to be detected Minimum Pk Width Minimum width of the peak as determined by the difference in wavelength between the higher of the two adjacent minima and the opposing intersection of that higher minimum level and the peak profile See the screen displayed below Peak Detect on Zoom Determines whether peaks are re assessed and tabulated when the user zooms into a region of the wavescan If off leaves the peak detection as determined on the un zoomed display Sort Peaks By Determines the sequence that peaks are reported by Can be Wavelength Peak Height or Peak Width Draw Peaks Switches display of peak cursors on and off These show vertical dashed lines displaying the measured peak height and horizontal dashed lines showing the peak width Pressing Cancel ignores the selection pressing OK accepts it Page 55 a avescan User Defined Feak Sample 2 4 wW ave Scan Parameters Print Abs T Peak Detection Delete Peak Graph Scale Sample Mumber Save
30. a m4 DLE gb 140 Le Ll4 Screen see below OR Press Back to return to the Standards Screen Results screen Step 14 Pipette on the reference sample and lower the sampling head If Auto Read is off press the OA 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 15 Clean the top and bottom plates pipette on the sample and lower the sampling head If Auto Read is off press FE The concentration of the sample is taken and displayed Repeat step 15 for all samples Press to return to the Protein Screen Press KRM to display available Options which are described below Options select using keypad numbers 1 Return to Parameters Screen step 1 above z os 2 Print result via selected method a E Graph 3 Toggle Graph on off Displays the calibration graph cursors give values for last measured sample 7 Sample Number add a prefix to the sample number and reset the incrementing number to the desired value o Sencie Nun 8 Save Method use the alpha numeric keys to enter a name ampie MUMDer seve Method for the method and press Save EP suto Print 9 Auto Print toggles Auto Print on off Esco Exit Options by pressing Or wait Version 4 0 Page 48 5 APPLICATIONS 5 1 File system o ef ef Single wavelength Concentration VAY aWESCan e 4 of Kinetic
31. a selected method Toggle Graph on off Displays calibration graph cursors give values for last measured sample Sample Number add a prefix to the sample number and reset the incrementing number to the desired value 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 Auto Print toggles Auto Print on off Ese or wait Page 63 5 7 Multiple Wavelength This makes up to 5 absorbance measurements on the same sample Note that the absorbencies measured are not nomalized to a 10mm pathlength so a maximum value of 2 5A is to be expected The procedure is as follows Multi wavelength Parameters Wavelengths E Pathlength Multi wavelength Parameters i E B ere if i 3 O Pao 300 320 340 360 380 400 420 Parameters Frint Sample Humber Save Method Subto F rim a 1 C Print Graph Version 4 0 Step 1 Select the number of Wavelengths range 2 5 Press the down arrow Step 2 Select Pathlength from 0 5mm or 0 2mm Press to go to the second Parameters Screen or Cancel Ese to return to the Applications Folder Step 3 Enter the first wavelength using either the number keys or the left and right arrows Press the down arrow Step 4 Enter the second wavelength as above and repeat for the number of wavelengths selected up to 5 Press OK to enter the Results Scr
32. al absorbencies are not nomalized to a 10mm pathlength The procedure is as follows Step 1 Enter the first Wavelength by using the keypad numbers or the left and right arrows Press the down arrow Absorbance R atio Wavelengths wavelength 1 Step 2 Enter the second Wavelength as above EE Press the down arrow 280 nm Step 3 Select whether Background Correction is to be applied to both Background wavelengths 1 and 2 using the left and right arrows Step 4 If Background Correction is On Enter the third wavelength from which the Background nex cance Correction will be obtained Step 5 Press Next to enter the Parameters Screen OR Press Cancel E to return to the Applications Screen Absorbance RA atio Parameters Pathlength Factor Absorbance Ratio Parameters Screen Step 6 SILT arto Select Pathlength from 0 5mm or 0 2mm Press the down arrow Step 7 Dilution Factor known Enter a dilution factor by using the keypad numbers range 1 00 9999 OR Step 7 Calculate Dilution Factor Press the options key RIKI M Enter the Volume of the sample range 0 01 9999 using the keypad numbers Press the down arrow Enter the volume of Diluent range 0 01 9999 using the keypad Dilution Factor i numbers Absorbance R atio Parameters Press OK to calculate the dilution factor and return to the Volume Parameters Screen or press Cancel to cancel selections Step 8 M
33. allowed totalling 81 methods 5 Utilities Instrument set up options and games Games only available if selected via the Preferences Screen 2 3 Data export An integral printer is available for the instrument this may be either supplied pre installed or as an optional accessory The installation procedure is described in section 8 1 Figure 2 NanoVue with Printer Data can be transferred to a PC either via a Bluetooth accessory supplied pre installed or as an optional accessory or viaa USB cable The software to perform this task PVC Print Via Computer is a small application running under Windows 2000 Windows XP or Windows Vista PVC can operate via USB and Bluetooth simultaneously PVC can store data either in a common directory or can be configured to save to independent directories by both file format and connection The data may be stored as an Excel spreadsheet an EMF graphics file a comma delimited csv data file a tab delimited txt data file or in native PVC format Some users may find it convenient to print through the PC directly to a printer already attached to it The software to enable this option will be found on the CD provided Version 4 0 Page 7 2 4 Sample treatment NanoVue uses a unique sampling head which enables users to accurately measure the optical characteristics of samples volumes as low as 0 5ul The sample is applied to a horizontal plate having a hydrophobic surface the
34. ampling head If Auto Read is off press MWL use C to clear previously stored results before measuring to measure the standard and store the result Repeat step 12 for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 Standards Manual selected When all standards are measured the OK box appears Press OK 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 Step 11 Standards selected Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed Step 12 Standards selected Press Replicates to display the replicate entry boxes Use C to clear previously stored results before measuring Clean the top and bottom plates pipette on the replicate standard and lower the sampling head If Auto Read is off press W to measure the standard and store the result Repeat for all replicates and standards Use Next ESS to bring up fields for the next standard A graph will display the results and the fitted curve as the measurements are inpu
35. an be heard buzzing at startup but there is no image on the screen then the display may be faulty Please contact your supplier Page 83 4 PO PO pO PO gt O gt O gt O PO pO PO 2O PO POD PO gt O Frequently asked questions How accurate is NanoVue Typically within 2 What sort of reproducibility should be produced with NanoVue Typically 0 005 A at low concentrations and for higher concentrations standard deviations are typically lt 2 Does NanoVue produce results for a continuous spectrum or just selected wavelengths lt produces results for a continuous spectrum from 200 1100 nm Do nucleic acids require purification before being measured Yes Absorbance measurements are not specific for a particular nucleic acid and will be affected by the presence of other molecules which absorb at 260 nm Is wiping the top and bottom plates adequate to prevent carryover Yes the hydrophobic surface of the plates are resistant to sample adherence ensuring dry tissue is effective in removing the sample If in doubt use a tissue wetted with water or a solution of a mild detergent see section 3 1 Step 6 for details Are there solvent restrictions Most common laboratory solvents including dilute acids may be used but they should be immediately wiped off the sample plate after measuring The NanoVue has been tested with methanol isopropanol 2 propanol and acetone though they are not recommended due to th
36. ary Function Keypad number Description 1i 1 Set correct date and time Date and Time 2 Select preferred language and number format Regional 3 Printer output options 4 Select screen layout themes and history Preferences 5 Adjust screen contrast amp brightness Contrast i 6 Shows serial number and software version Aout 7 Pathlength Calibration Frathlenath Calibration Re 8 Spectro Blocks and Sudoku Games Version 4 0 Page 68 7 1 Date and Time The procedure is as follows Date and Time Day Hour Month Minute Wear 7 2 Regional Sets Language and Number Format Regional Language Humber Format 9399 93 7 3 Printer Sets up printing options Printer Auto Print Printer Version 4 0 Enter the Day using the keypad numbers or left and right arrows Press the down arrow Enter the Month Press the down arrow Enter the Year Press the down arrow Enter the Hour Press the down arrow Enter the Minute Seconds are zeroed when OK is pressed Press OK to store the settings and return to the Utilities Screen OR Press Cancel to return to the Utilities Screen without storing the time The procedure is as follows Select a Language Options are English French Spanish ltalian or Japanese Press the down arrow Set the decimal point style Options are or Press OK to store the settings and return to the Utilities Screen OR
37. ase_type base n Version 4 0 Page 25 Conversion factor The Conversion Factor is given by Molecular weight agcpe 2 Eascpe where Eascoe 2 x Eag Egc Ecp Epe Eg Ec Ep e The molecular weight MW of a DNA oligonucleotide is calculated from MW g mole dA x 312 2 dC x 288 2 dG x 328 2 dT x 303 2 MW counter ion x length of oligo in bases for RNA oligonucleotide dT x 303 2 is replaced by dU x 298 2 The MW calculated using this equation must be adjusted for the contribution of the atoms at the 5 and 3 ends of the oligo For phosphorylated oligos add 17 2 x MW of the counter ion For non phosphorylated oligos subtract 61 MW of the counter ion The MW g mole of the most common oligo counter ions are Na sodium 23 0 K potassium 39 1 TEA triethylammonium 102 2 Other Defaults to 1 0 H Variable 0 1 999 9 in next option box Calculated molecular weight a weight is added for each base looked up from a table The weight of the counter ion is added for every base from a small table for the known ions If phosphorylated then the system adds 17 0 plus two counter ions otherwise it subtracts 61 0 and one ion Theoretical Absorbance for each adjacent pair of bases nearest neighbors a weight is accumulated using a table For each internal base a weight is subtracted using another table Separate tables are used for DNA and RNA Calculated factor this is just
38. be repeated if a poor reading has been obtained Use C to clear the previous reading Step 12 Press OK to accept the calibration and go to the Results Screen see below OR Press Back to return to the Standards Screen Page 61 Standard Curve Calibration Calibration Screen Replicates on A This shows the calibration values and allows standards to be Le oe fo measured ae Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the i sample will need to be replaced and the 0A 100 T key pressed MM again ld 20 40 40 g0 BO Step 11 sa Press Replicates ES to display the replicate entry boxes Use C to clear previously stored results before measuring Clean the top and bottom plates pipette on the replicate standard and lower the sampling head If Auto Read is off press AS to measure the standard and store the result Repeat for all replicates Press Next to measure the next standard and repeat the process 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 Step 12 Press Next to accept the calibration and go to the Results Screen see below OR Press Back to return to th
39. 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 0 to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters or Cancel E Step 8 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 Step 9 Select Pathlength from 0 5mm or 0 2mm Step 10 Press OK to enter the Results Screen OR Press Back to return to the Parameters 1 Screen Results Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Clean the top and bottom plates pipette on the sample and lower the sampling head If Auto Read is off press FEL 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 Semele Eee eee The table below the graph gives absorbance values at To start of calculation T finish of calculation change in absorbance slope regression parameter R of the calculated slope and the
40. creen Run the standard by pressing t OR Press cancel to return to the Measure Screen Step 9 Clean the top and bottom plates pipette on the sample and lower the sampling head If Auto Read is off press FE The concentration of the sample is displayed Results shown as indicate that the concentration is out of range Repeat step 8 for all samples Press to return to the Applications Screen Press KHK to display available Options which are described below Options select using keypad numbers 1 Return to Parameters Screen step 1 above E Parameters 2 Print result via selected method E Frin 3 Toggles on off displaying a graph of wavescan 20nm E Graph from selected wavelength Fun Standard 4 Return to Run Standard Screen 7 Sample Number add a prefix to the sample number and reset the incrementing number to the desired value E Sample Number 8 Save Method use the left and right arrows to select a CP Save Method folder to store in Favourites Methods 1 9 press the down E Auto Print y arrow and enter name 9 Auto Print toggles Auto Print on off Exit Options by pressing or wait Version 4 0 Page 53 5 4 Wavescan An absorption spectrum can be obtained from NanoVue enabling simple identification of peak height and position Note that the values reported are NOT nomalized to a 10mm pathlength so if comparison is being made to other systems the a
41. crementing number to the desired value nS 8 Save Method use the left and right arrows to select a folder Sample Number to store in Favourites Methods 1 9 press the down arrow E Save Method and enter name E Auto Print wi 9 Auto Print toggles Auto Print on off Exit Options by pressing or wait Version 4 0 Page 66 6 FAVOURITES AND METHODS These folders are the storage locations for any user modified Applications Methods that are saved in the Options menu Both are accessible from the opening NanoVue Screen Favourites Folder This location should be used for any frequently used methods as it gives immediate access from the top level display to up to 9 methods by one button press Methods Folder This contains a further 9 storage folders also accessible from the top level display folder and within each may be stored 9 methods thus allowing a total of 81 methods accessible by two button presses To save a method the procedure is Step 1 From the Results Screen of any application press the options JVI button Single wavelength Step 2 Select option 8 Save Method or press the number 8 directly from the Results Screen Folder Step 3 Select the folder in which to store the method Options are Methods 1 9 or Favourites Method Name Press the down arrow Step 4 Use the alohanumeric keys to enter a name for this method Save Method Press OK to store the method and return to the R
42. ds Manual selected 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 backspaces and clears the last digit entered Step 10 Standards Manual selected Press Next to enter the Calibration Screen If there are duplicate or non monotonic increasing entries the unit will beep and highlight the incorrect entry OR Bradford Standards Press Back to return to the Parameters Screen Version 4 0 Page 40 Bradford Calibration Bradford Calibration Standards 0 200 0 400 0 600 0 400 0 463 A 1 000 1 400 0 055 4 0 196 4 0 337 A 0 592 4 0 7194 Bradford Calibration Replicates T 0 058 4 0 055 4 0 056 A Bradford Calibration Standards 0 700 0 400 0 600 0 400 0 463 A 1 000 1 400 0 055 4 0 1956 A 0 337 4 0 592 4 0 7194 Version 4 0 ia E 14 DLE OF 140 Le L4 0 4 y r E a 2 AH Ob OB LO Le 14 D Ly D La D 10 D OG nts 14 GE mE LO LE LY 0 4 na 14 DLE OB LO Le L4 Calibration Screen Replicates off This shows the calibration values and allows standards to be measured or entered using the keypad numbers if calibration mode is manual Step 11 Standards selected Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples unti
43. e Standards Standard Curve Calibration 25 Standard Curve Calibration Standards 0 14 10 0 ooz74 aa 0 0604 Am 0 097 A 0 08 0 133 4 20 0 31 0 0 05 0 04 i3 mm 3o u0 5O E a 5 1 w i on io 20 30 u0 SO 50 Version 4 0 Standards Screen Step 8 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 Step 9 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 This shows the calibration values and allows standards to be measured Step 10 Pipette on the reference sample and lower the head If Auto Read is off press the OA 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 11 Clean the top and bottom plates pipette on the standard and lower the sampling head use C to clear previously stored results to measure before measuring If Auto Read is off press 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
44. e Standards Screen Calibration Manual entry Standard Curve Calibration 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 Standards 10 0 0 075 4 270 0 0 045 A 31 0 O 082 A 627 0 0 115 A 0 06 D 04 Press OK to accept the calibration and go to the Results E Screen see below DOG io a3 am o so E OR Renicates eak Press Back to return to the Standards Screen Results Screen Step 13 Sample Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed ad au Clean the top and bottom plates pipette on the sample and Standard Curve lower the head If Auto Read is off press AEE The concentration of the sample is taken and displayed Repeat step 14 for all samples Press to return to the Applications Screen Version 4 0 Page 62 Parameters Frrint Graph OOS Sample Mumber Save Method Subo Frint vl 8 Version 4 0 Options select using keypad numbers 1 2i 3 Exit Options by pressing Return to Parameters Screen step 1 above Print result vi
45. e that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters or Cancel E Step 7 To enter the Results Screen with the selected parameters press Cancel the selections and return to the Applications Screen by pressing Cancel E Step 8 if using a Factor Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 9 Clean the top and bottom plates pipette on the sample and lower the head If Auto Read is off press FE The concentration of the sample is displayed Results shown as indicate the concentration is out of range Repeat step 7 for all samples Page 52 Press to return to the Applications Folder Press KHK to display available Options which are described below Concentration lf Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Concentration nun Sania Step 8 if using Standard mode Pipette on the reference sample and lower the sampling head Press Halll to display the Run Standard S
46. e that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters or Cancel E Step 5 Enter the Pathlength Options are 0 5mm or 0 2mm Press Next to move on to the next parameters screen or Ese Cancel to return to the Protein Screen Step 6 Enter the type of Curve Fit Options are Regression straight line Zero Regression forces the straight line through the origin Interpolated or Cubic Spline Press the down arrow BCA Parameters Step 7 Slat Select the Calibration Mode either Standards measure ReneS prepared standards Manual keypad data entry go to step 9 or New Standards means new standards are measured each Calibration time the method is used Standards Step 8 only if Standards is selected Select the number of Replicates using the left and right arrows Replicates This determines the number of standards to be measured and averaged at each standard concentration point Can be Off 2 or 3 Press Next to enter the Standards Screen OR Press Cancel to cancel selections and return to the Standards Protein Screen CA Step 9 Standards Manual selected aye e 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 backspaces
47. ealthcare s website is http www gehealthcare com or http www gehealthcare com worldwide htm Support agreements that help you to fulfil the demands of regulatory guidelines concerning GLP GMP are available Calibration certification Certificated engineers and calibrated test equipment Approved to ISO 9001 standard Choice of agreement apart from break down coverage can include Preventative maintenance Certification Accessories Built in Printer accessory 28 9182 27 Spare paper for printer 20 rolls 28 9182 26 Bluetooth accessory 28 9182 25 Sample Plate Replacement kit 28 9244 06 Pathlength Calibration Kit 28 9244 05 PVC software 28 9231 88 Version 4 0 Page 79 9 0 TROUBLESHOOTING 9 1 Pathlength Calibration Over range Under exceptional circumstances an error message may be given when undertaking a pathlength calibration because one or both of the pathlengths are too far from 0 2 or 0 5mm for the instrument to Calibration failed because the compensate This means that the pathlengths will need manually calculated pathlengths are out of adjusting This can be done within the Pathlength Calibration function as described in section 7 7 Step 1 Enter the certificate ID on the certificate bottle of the calibration solution using the keypad Press the down arrow Step 2 Enter the calibration value of the calibration solution Note this down and divide by two to give the target value for the 0 5mm pathleng
48. ear it buzz at start up Is the power supply securely connected to the instrument Turn off the instrument disconnect and reconnect the power supply and then turn on the instrument If the problem persists please contact your supplier Check that you are using the correct power supply It should be an 18V unit Make sure the sample plates are clean have been fitted securely and are well seated Is the xenon source firing when you press TT Can you hear it buzz at start up Is the power supply securely connected to the instrument Turn off the instrument disconnect and reconnect the power supply and then turn on the instrument lf the problem persists please contact your supplier Page 81 What is it Calibration problem Possible lamp failure no light Low light on reference channel No light on reference channel Calibration problem Cell holder obstructed UV IR What should you do Check that you are using the correct power supply It should be an 18V unit Make sure the sample plates are clean have been fitted securely and are well seated Make sure any packing pieces have been removed form the plates Is the xenon source firing when you press Can you hear it buzz at start up Is the power supply securely connected to the instrument Turn off the instrument disconnect and reconnect the power supply and then turn on the instrument If the problem persists please contact your supplier Check that you
49. een OR Press Cancel to return to the Applications Screen Step 5 Pipette on the reference sample and lower the head If Auto Read is off press the OA 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 6 Clean the top and bottom plates pipette on the sample and lower the head If Auto Read is off press Repeat step 6 for all samples Results A scan plot covering the range of wavelengths selected with cursors at the relevant wavelengths and a table of values is displayed Press to return to the Applications Screen Press KHK to display available Options which are described below 1 Return to Parameters Screen step 1 above 2 Print result via selected method 4 Print Graph using selected method Grayed out if no data are available 7 Sample Number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save Method use the left and right arrows to select a folder to store in Favourites Methods 1 9 press the down arrow and enter name 9 Auto Print toggles Auto Print on off Exit Options by pressing or wait Page 64 5 8 Absorbance Ratio This makes 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 Note that the individu
50. eir high volatility How do check the accuracy of NanoVue Do a calibration following the instructions in section 7 7 How often should NanoVue be calibrated At least every 6 months or at a frequency determined by the standard operating procedures of your laboratory A calibration should also be performed if the sample plates are changed What kind of light source does the NanoVue use A pulsed xenon flash lamp How often will the lamp require changing The lamp should last for several years of use If it does need changing this should be done by a service engineer from your supplier Is the flash lamp on continuously or only when performing a measurement The lamp is only on when taking measurements Does NanoVue require a computer to operate No NanoVue operates as a stand alone instrument although it can be connected to a PC through the USB port to enable connection to a remote printer LAN or electronic data archive Is it possible to connect a USB memory stick to the USB port on the NanoVue No but export of data via a USB lead or via a Bluetooth wireless link is supported What volume of sample can use with the NanoVue On the 0 2mm pathlength volumes as low as 0 5ul can be used For Auto or 0 5mm use 2ul Do not use volumes greater than 5ul as this may spread too far over the plates My droplet is spreading out over the lower plate and get inconsistent readings Either the plates have picked up some contamination or t
51. en The Result at the selected wavelength is displayed on the screen Use the left and right arrows to move the cursor and display the value at the cursor position 15nm from set wavelength Press Cancel to return to the Applications Screen Press KRM to display available Options which are described below Page 50 DOS S806 Parameters Frint Abst 36 T Print Graph Sample Humber Save Method Awuto Print a Version 4 0 Options select using keypad numbers Return to Parameters Screen step 1 above Print result via selected method Toggle between Absorbance and T mode Print Graph greyed out if no data are available Sample Number add a prefix to the sample number and reset the incrementing number to the desired value 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 oS I Oo Exit Options by pressing or wait Page 51 5 3 Concentration This makes concentration measurements on samples by measuring the amount of light that has passed through a sample relative to a reference this can be air Concentration is obtained by multiplying the measured absorbance at a specific wavelength by a factor Note that the system does not nomalize the result to a 10mm pathlength so the factor may need to be modified accordingly if brought over from anoth
52. eplaced and the 0A 100 T key pressed again Step 12 Standards selected Press replicates to display the replicate entry boxes Use C to clear previously stored results before measuring Clean the top and bottom plates pipette on the replicate standard and lower the sampling head If Auto Read is off press aig E to measure the standard and store the result Repeat for all replicates and standards Press Next to move from replicates of one standard to replicates of the next standard A graph will display the results and the fitted curve as the measurements are input Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 Standards Manual selected Press OK to accept the calibration and go to the Results Screen see below OR Press Back to return to the Standards Screen Page 47 Biuret Calibration Calibration Manual entry 0 349 4 gora A o Shows previously entered calibration values and allows values to 0 146 A ra be entered via the keypad The highlighted box can be edited in ozsa E Z 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 Oda7 a oa 0 542 4 Press OK to accept the calibration and go to the Results Oot L
53. er system The factor may also be calculated by the instrument by measuring a standard of Known concentration The procedure is as follows Concentration Parameters ELIS wavelength Hode Concentration Parameters Version 4 0 Step 1 Select Pathlength from 0 5mm or 0 2mm Step 2 Set Wavelength by using keypad numbers or left and right arrows range 190 1100nm Press the down arrow key Step 3 Select the Mode Factor user entered or Standard factor is calculated from a calibration sample using the left and right arrows Press the down arrow key Step 4 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 Press the down arrow key The factor should be modified to include a correction for the selected pathlength if needed Step 5 if Standard is selected Enter the Concentration using keypad numbers range 0 01 9999 Use the C button to delete the last digit entered Press the down arrow key Step 6 Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Options key kk and then use the left right arrows to select from ug ml ug ul pmol ul mg dl mmol l umol l g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed decimal points DP to be selected from 0 to 2 Not
54. es Games only available if selected via the Preferences Screen Page 11 3 OPERATION AND MAINTENANCE 3 1 Sample application guide Step 1 Lift the sampling head to the vertical position and using a low volume 0 10 ul pipette take up approximately 2ul of sample A reliable pipette and matching high quality tips are strongly recommended When removing a very small aliquot from a larger volume ensure that the sample is truly representative Filtration may also be advantageous Step 2 Carefully apply the sample so that it sits over the black spot between the four alignment spots See Figure 4 Take care not to introduce bubbles into the sample After the plunger is depressed lift the pipette up carefully so as not to drag the droplet off target Figure 4 Pipette sample here Figure 5 Version 4 0 Page 12 Step 3 Observe the position and shape of the drop it should resemble the one shown in Figure 6a rather than 6b A noticeably spread out sample indicates the target area may be contaminated and requires cleaning see below Gently lower the sampling head Figure 6a Figure 6b Step 4 If a program has been selected and the Auto Read function is set to On see section 2 6 the sample reading process will begin automatically a reference scan will be taken first followed by the measurement scan If the Auto Read function is set to Off use the 0A 100 T and EWT buttons to take the reference and measureme
55. es and standards Press Next to move from replicates of one standard to replicates of the next standard A graph will display the results and the fitted curve as the measurements are input Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 Standards Manual selected Press OK to accept the calibration and go to the Results Screen see below OR Press Back to return to the Standards Screen Page 41 Bradford Calibration Standards 0 200 0 400 0 600 0 800 1 000 1 400 wavelength 535 nm Absorbance 0 059 4 0 196 A 0 337 A 0 592 A ormsa ME ri 2 p d 1 0 2 0 4 16 0 8 1 0 l z 14 Bradford Concentration 4 244 Curve Fit Regession OO DoS Version 4 0 Parameters F rint Graph Sample Mumber Save Method Subo Frint 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 Pressing the down arrow from the last standard will bring up the OK box Press OK to accept the calibration and go to the Results Screen see below OR Pre
56. es the number of standards to be measured and Biada averaged at each standard concentration point Can be Off 2 or 3 Press Next to enter the Standards Screen OR Press Cancel to cancel selections and return to the Protein Screen Lowry Standards Standards Screen Step 9 Standards Manual selected 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 backspaces and clears the last digit entered Step 10 Standards Manual selected Press Next to enter the Calibration Screen If there are duplicate or non monotonic increasing entries the unit will beep and highlight the incorrect entry OR Press Back to return to the Parameters Screen Version 4 0 Page 43 Lowry Calibration OOS ne O14 G6 OF LO Le 14 Lowry Calibration Standards 0 100 0 077 A 0 400 0 600 0 600 0 132 4 0 056 A a e in 0 054 A 0 10 1 000 0 1694 1 400 0 205 4 he O14 O65 OF LO Le L14 Lowry Calibration Replicates 1 O01 A O01 A O01 A man O 017 A mos he O14 O65 OF LO Le 14 Lowry Calibration Standards 0 100 0 400 0 077 A 0 056 A a e in 0 600 0 095 4 0 132 4 a e 0 800 1 000 1 400 0 169 0 205 A he O14 O16 O8 LO Le L14 Version 4 0 Calibration Screen Replicates off This shows the calibration values and allows standards to
57. esults Screen OR Press Cancel to return to the Results Screen without storing the settings Saved Methods can be locked unlocked and deleted using the Options menu Go to the relevant folder by selecting either option 3 Favourites or option 4 Methods from the opening NanoVue Screen Select the method you wish to lock unlock or delete by pressing the relevant keypad number and then press the KIM key Delete Method Press 1 to select Delete Method select the Method to be deleted using the left and right arrows Press to delete the method OR cancel to return to Favourites Methods Screen Lock Method 0 Press 2 to select Lock Method Single wavelength my method 1 Select the Method to be locked using the left and right arrows Press the down arrow Select a Pass Code using the keypad numbers or left and right arrows Press to lock the Method OR cancel E to return to the 2 neee Menot Favourites Methods Screen P Unlock Method Unlock Method Press 3 to select Unlock Method Select the Method to be unlocked using the left and right arrows Press the down arrow Enter the Pass Code using the keypad numbers or left and right arrows Press to unlock the method OR cancel to return to the Favourites Methods Screens Version 4 0 Page 67 7 UTILITIES Press 5 to enter the utilities folder of e ef Oate and Time Regional Printer OG of Contrast About Fathlength Calibration Summ
58. etic compatibility generic emission standard electrical equipment for measurement control and laboratory use Electromagnetic compatibility generic immunity standard part 1 Residential commercial and light industry This appliance is marked according to the European directive 2002 96 EC on Waste Electrical and Electronic Equipment WEEE By ensuring this product is disposed of correctly you will help prevent potential negative consequences for the environment and human health which could otherwise be caused by inappropriate waste handling of this product The symbol K on the product or on the documents accompanying the product indicates that it may not be treated as household waste Instead it should be handed over to the applicable collection point for the recycling of electrical and electronic equipment Disposal must be carried out in accordance with local environmental regulations for waste disposal This device is currently exempt from the European RoHS requirements as it falls under category 9 of the Directive This device does NOT fall under any categories detailed in the Chinese EIP classification list dated 16 3 2006 but for informational purposes only is currently non compliant with Chinese RoHS requirements as it contains lead based solders but is safe for 20 years of use Page 5 2 INTRODUCTION 2 1 Your NanoVue NanoVue is a simple to use UV Visible instrument with twin CCD array detectors 1024 pixels and few m
59. f of ef Bradford Lowry Biuret Version 4 0 Page 16 4 2 Nucleic Acids 4 2 1 Theory Nucleic acids can be quantified at 260nm because at this wavelength there is a clearly defined peak maximum A 50ug ml DNA solution a 40 ug ml RNA solution and 33ug ml solution of a typical synthetic Oligonucleotide all have an optical density of 1 0A ina 10mm pathlength cell These factors 50 40 and 33 respectively can be inserted into the formula 1 below although they do vary with base composition and this can be calculated more precisely if the base sequence is known 1 Concentration Abs260 Factor where means multiplied by NanoVue will default to factors 50 for double stranded DNA 40 for RNA and 33 for single stranded DNA and Oligonucleotides It also allows manual compensation for dilution aided by a dilution calculator Nucleic acids extracted from cells are accompanied by protein and extensive purification is required to remove the protein impurity The 260 280nm absorbance ratio gives an indication of purity however it is only an indication and not a definitive assessment Pure DNA and RNA preparations have expected ratios of 1 7 1 9 and gt 2 0 respectively Deviations from this indicate the presence of impurity in the sample but care must be taken in the interpretation of results The 260nm reading is taken near the top of a broad peak in the absorbance spectrum for nucleic acids whereas the 280nm reading naturally occ
60. ference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 9 Clean the top and bottom plates pipette on the sample and lower the sampling head If Auto Read is off press MWE This measures at the selected wavelengths and displays the results The ratio of the absorbance values at wavelengths 1 and 2 are calculated The Concentration is based on the absorbance at wavelength 1 Repeat step 9 for all samples Press to return to the Life Science Screen Press KHK to display available Options which are described below Version 4 0 Page 19 OO tolon Version 4 0 Parameters F rint Graph Sample Mumber Save Method Subo Frint Options select using keypad numbers Return to Parameters Screen step 1 above Print result via selected method Toggle Graph on off The graph shows a Wavescan plot across the range 220nm to 320nm with cursors denoting 230 260 280 and if Background Correction selected 320nm Sample Number add a prefix to the sample number and reset the incrementing number to the desired value Save Method use the alpha numeric keys to enter a name for the method and press Save Auto Print toggles Auto Print on off Exit Options by pressing or wait Page 20 4 2 3 RNA meas
61. for the method and press Save 9 Auto Print toggles Auto Print on off Exit Options by pressing or wait Page 34 4 3 3 Protein A280 i Step 1 A Press 2 to select Protein A280 Mode Background Step 2 Select the Mode Options are Christian Warburg BSA IgG Lysozyme Molar extinction Mass extinction E 1 Pathi i Step 3 athlength Units g Select Pathlength using the left and right arrows Options are 0 5mm 0 2mm or Automatic Press the down arrow Dilution Factor Step 4 Dilution Factor known Enter the Dilution Factor using the keypad numbers range 1 000 to 9999 Use the C button to backspace and clear the last digit entered OR Step 4 calculate Dilution Factor PIAN ee ere Press KIEJM to enter the Dilution Factor Screen shown to the Dilution Factor left i Enter the Volume of the sample using the keypad numbers ene range 0 001 to 9999 Press the down arrow Enter the volume of the Diluent using the keypad numbers Diluent range 0 001 to 9999 Press OK to calculate the Dilution Factor and return to the Parameters Screen OR Press to cancel the selections and return to the Parameters Screen Step 5 Select whether Background Correction is to be used or not with the left and right arrows Protein 42780 Parameters Press the down arrow Step 6 Mode Christian Warburg BSA IgG Lysozyme alaia Panon Select the Units of measurement using the left and right arrows oa
62. hey may need to be changed See Section 3 Version 4 0 Page 84 10 Specification Wavelength range Monochromator Wavelength calibration Wavelength accuracy Wavelength reproducibility Light sources Detector Photometric range Photometric accuracy Digital output Dimensions Weight Power input SPECIFICATION AND WARRANTY 200 1100 nm scanning 200 950 nm Flat grating Automatic upon switch on 2 nm across range 1 nm from 240nm to 330nm 0 5 nm Pulsed xenon lamp 1024 element CCD array O to 125 A 10mm pathlength equivalence 1 at 257nm at 0 7 0 8A USB port standard Bluetooth option 260 x 390 x 100 mm lt 4 5 kg 18Vdc from a 90 250 V 50 60 Hz Max 30 VA mains power pack Specifications are measured after the instrument has warmed up at a constant ambient temperature and are typical of a production unit As part of our policy of continuous development we reserve the right to alter specifications without notice Warranty GE Healthcare 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 GE Healthcare can accept no liability for loss or damage however caused arising from the faulty or incorrect use of this product The lamp is warranted for 3 years if the product has been used according to the instructi
63. in DAWA Yield Step 1 5 Clean the top and bottom plates pipette on the sample and Nucleotides lower the sampling head If Auto Read is off press AME The 20 7 system returns the dye concentration Oye Per Probe TE Repeat step 15 for all samples Press _ to return to the Life Science Screen Press KKM to display available Options which are described below EP Psrameters Note that very high dye concentrations can leave visible E Print deposits on the sample plates This can easily be removed with Graph a gentle rub with ethanol C Results y Options select using keypad numbers ee 1 Return to Parameters Screen step 1 above Semple Number 2 Print result via selected method E Save pence 3 Toggle Graph on off The graph shows the spectral scan E avtoPrint of the dye sample over the wavelength range of interest 4 Toggle Results on off When off the results screen shows the absorbance values and absorbance ratios When on see picture to left the results screen shows the total DNA DNA Yield nucleotides and number of dyes probe This only functions if the graph display is off 7 Sample Number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save Method use the alpha numeric keys to enter a name for the method and press Save 58 5 9 Auto Print toggles Auto Print on off Exit options by pressing or wait AZEBOSA2ZEO 4 795 Adyel
64. inverse of the 10mm absorbance of a 1 solution of the protein under test Version 4 0 Page 31 Rapid measurements such as this at Abs280 are particularly useful after the isolation of proteins and peptides from mixtures using spin and Hilrap columns by centrifuge and gravity respectively Protein Determination at 595 546 562 and 750nm 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 595nm this is usually BSA bovine serum albumin The Biuret method depends on reaction between cupric ions and amino acid residues in an alkali solution resulting in the formation of a complex absorbing at 546nm The BCA method also depends on reaction between cupric ions and amino acid residues but in addition combines this reaction with the enhancement of cuprous ion detection using bicinchoninic acid BCA as a ligand giving an absorbance maximum at 562nm The BCA process is less sensitive to the presence of detergents used to break down cell walls The Lowry method depends on quantifying the color obtained from the reaction of Folin Ciocalteu phenol reagent with the Tyrosyl residues of an unknown protein and comparing with those derived from a standard curve of a standard protein at 750nm this is usually BSA Detailed protocols are supplied with these assay kits and must be closely followed to
65. ity Oligonucleotide concentration Tm Calculation Cy Dye concentration and a variety of protein measurements In all of these applications instead of the actual absorbance measurement being used the value is normalized to reflect a standard pathlength of 10mm so that generally accepted factors 50 for DNA 40 for RNA and 33 for proteins for example can be used to calculate concentration As an example if the absolute absorbance of the sample is 0 025A using a pathlength of 0 5mm the normalized absorbance shown will be 0 500A NanoVue is therefore ideally suited to the life scientist where sample is limited and speed and convenience of analysis is key Version 4 0 Page 6 2 2 File system After switch on and automatic checking the instrument defaults to a home page entitled NanoVue This page displays five folders which form the topmost layer of a simple file tree which is the basis of the user interface The folder screens are reached by pressing the appropriate number on the keypad with return to the top level by means of the Esc key The folders group various facilities together as follows 1 Life Science Standard life science applications such as nucleic acid and protein assays 2 Applications General spectroscopic applications Favourites A folder to store your more frequently used methods Inactive when empty 4 Methods Contains 9 folders that can store less frequently used methods Up to 9 methods per folder are
66. l changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 12 Standards selected Clean the top and bottom plates pipette on the standard and lower the sampling head If Auto Read is off press MWE use C to clear previously stored results before measuring to measure the standard and store the result Repeat step 12 for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 Standards Manual selected When all standards are measured the OK box appears Press OK 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 Step 11 Standards selected Insert the reference sample Press the 0A 100 key This will be used for all subsequent samples until changed Step 12 Standards selected Press replicates to display the replicate entry boxes Use C to clear previously stored results before measuring Clean the top and bottom plates pipette on the replicate standard and lower the sampling head If Auto Read is off press ME to measure the standard and store the result Repeat for all replicat
67. libration Standards Replicates Biuret Standards Version 4 0 Step 1 Press 6 to select Biuret method Step 2 The Wavelength for this method is fixed at 546nm Step 3 Enter the number of Standards 1 9 to be used in the curve using the keypad numbers or left and right arrows Press the down arrow Step 4 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 Kk and then use the left right arrows to select from ug ml ug l pmol ul mg dl mmol l umol 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 0 to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters or Cancel E Step 5 Enter the Pathlength Options are 0 5mm or 0 2mm Press Next to move on to the next parameters screen or Ese Cancel to return to the Proteins Folder Step 6 Enter the type of curve fit Options are Regression straight line Zero Regression forces the straight line through the origin Interpolated or Cubic Spline Press the down arrow Step 7 Select the calibration mode either Standards measure prepared standards Manual keypad data entry g
68. ly and are well seated Is the xenon source firing when you press TT Can you hear it buzz at start up Is the power supply securely connected to the instrument Turn off the instrument disconnect and reconnect the power supply and then turn on the instrument lf the problem persists please contact your supplier Check that you are using the correct power supply It should be an 18V unit Make sure the sample plates are clean have been fitted securely and are well seated Is the xenon source firing when you press 1 Can you hear it buzz at start up Is the power supply securely connected to the instrument Turn off the instrument disconnect and reconnect the power supply and then turn on the instrument If the problem persists please contact your supplier Check that you are using the correct power supply It should be an 18V unit Make sure the sample plates are clean have been fitted securely and are well seated Is the xenon source firing when you press Can you hear it buzz at start up Is the power supply securely connected to the instrument Turn off the instrument disconnect and reconnect the power supply and then turn on the instrument If the problem persists please contact your supplier Check that you are using the correct power supply It should be an 18V unit Make sure the sample plates are clean have been fitted securely and are well seated Is the xenon source firing when you press 1 Can you h
69. n Use the cursors to select the square and the keypad to enter a number Invalid numbers cannot be entered Cells can be locked or unlocked by using the decimal point Unlocked cells can be cleared using the C key see also Option key below The user mode starts with a blank grid Sudoku Setup Sudoku Game 1 Hode Computer Game Options Press KHK to display the Options Screen Return to the Setup Screen The instrument solves the game for you Clear all entries Save the game Use the left and right arrows to select a folder to store the game in Favourites Methods 1 9 press the down arrow and enter a name a ae Press Cancel to return to the Utilities Screen Version 4 0 Page 74 8 ACCESSORIES INSTALLATION The user can install either a printer or a Bluetooth module 8 1 Printer installation Step 1 Remove the power cable and turn the instrument over onto a soft surface taking care not to damage the sampling head Release the outermost screws using the Allen key provided Figure 12 Step 2 Turn the instrument back over and remove the accessory Covers Figure 13 Step 3 Attach the printer cable Version 4 0 Page 75 Figure 14 Figure 16 Step 6 Switch the instrument on and go to utilities instrument preferences and select the Built in printer Figure 17 Version 4 0 Page 76 Step 4 Lower the printer onto the locating bosses Step 5
70. n Factor using the keypad numbers range 1 00 to 9999 Use the C button to backspace and clear the last digit entered OR seta Step 8 calculate Dilution Factor Dilution Factor f Press Kkr to enter the Dilution Factor Screen see third image to the left Enter the Volume of the sample using the keypad numbers range 0 01 to 9999 Press the down arrow Enter the volume of the Diluent using the keypad numbers range 0 01 to 9999 Diluent Press OK to calculate the Dilution Factor and return to the Parameters Screen OR Press to cancel the selections and return to the Parameters Screen Version 4 0 Page 29 Step 9 Cy Dye Settings Enter the Conversion Factor using the keypad numbers Press the down arrow This defaults to 50 for cDNA but should be 40 Enter the Volume of the probe in ul Factor Background Step 11 Step 10 TEE Enter the amount of starting RNA in ng maximum 99 999 ng Step 12 if Background Correction is on Enter the Background Correction wavelength if required Step 13 Press OK to select these settings and start to measure OR press Back Ese to cancel the settings and return to the Parameters Screen Results Screen Step 14 Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on ry the sample will need to be replaced and the 0A 100 T key pressed aga
71. n or Cancel E to return to the Utilities Screen Pressing OK will show the next screen as left Step 3 Open the NanoVue sampling head and pipette 2 5ul of distilled water onto the correct position on the sample plate To reduce small errors being introduced due to droplet misplacement the higher volume is recommended Close the sampling head and press the Pathlength Calibration Calibration OA 1 OO T Key 05mm 02mm NanoVue will take a reference at both 0 2 and 0 5mm pathlengths 0 487 A This takes longer than usual in order to get the best quality answer 0 495 A The QA function is automatically switched on during this process oe This means that the instrument will require a further reference A sample to be measured to ensure the quality of that measurement aces The process will repeat until two successive measurements are in 0 492 4 agreement The sampling head will need to be lifted and the droplet 0 451 A replaced between measurements before the OA 100 T key 0 491 A becomes available When the instrument stops asking for further 0 494 A references proceed to Step 4 oK Step 4 The Calibration process now requires 10 measurements of the oT calibration fluid to construct a statistically valid result The Pathlength Calibration A ji consistency of the results can be seen as the table is populated Deviations of more than about 2 will cause an error and require further measurements ee f Open the sampling head clean off the water f
72. n to the Protein Screen Press KHK to display available Options which are described below Options select using keypad numbers E Fsrameters 1 Return to Parameters Screen step 1 above Print 2 Print result via selected method P Graph 3 Toggle Graph on off The graph shows a wavescan plot across the range 250nm to 330nm 7 Sample Number add a prefix to the sample number and reset the incrementing number to the desired value E Sample Number 8 Save Method use the alpha numeric keys to enter a E Save Method name for the method and press Save E Auto Print 9 Auto Print toggles Auto Print on off Exit Options by pressing or wait Version 4 0 Page 36 4 3 4 BCA The procedure is as follows Step 1 Press 3 to select BCA mode BCA Parameters Step 2 The Wavelength for this method is fixed at 562nm Step 3 Enter the number of Standards 1 9 to be used in the curve using the keypad numbers or left and right arrows Press the down arrow Step 4 see second image 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 kk and then use the left right arrows to select from ug ml Ug ul pmol ul mg dl mmol l umol 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 0 to 2 Not
73. nt scans respectively Note It is not recommended to raise and lower the head repeatedly to take multiple measurements of one sample this can cause the droplet to disperse If repeat measurements are required use the button Step 5 If you wish to keep the sample it can be recovered after the reading has been taken using a pipette Step 6 After taking the reading both the top and bottom plates should be cleaned by wiping away the sample using a soft lint free tissue Wipe the bottom plate towards you and the top plate upwards to avoid contaminating the return light path to the rear Version 4 0 Page 13 lt may be necessary to remove sticky or dried on residues Water or a dilute 2 detergent solution should suffice After using detergent the plates should be wiped a second time with either water or Isopropanol 2 propanol Figure 7a Figure 7b 3 2 Sample plates replacement lf after careful cleaning the droplet shape is still unsatisfactory or if either of the plates becomes damaged or there is deterioration of the hydrophobic coating then it will be necessary to replace them as a pair The glass plates are permanently fixed within plastic housings which clip to the sample handling module Replacements are available from your supplier Exchanging the sample plates is simple See Figures 8a and 8b They are available from your supplier under part number 28 9244 06 1 Remove the top sample plate Put one thumb at the
74. nt on off Ese or wait Page 22 4 2 4 Oligonucleotide measurement The procedure is as follows Step 1 Oligo Parameters Press 3 to select Oligo mode Step 2 Pathlensth Units Select Pathlength using the left and right arrows Options are 0 5mm 0 2mm or Automatic Press the down arrow Step 3 Dilution Factor known Dilution Factor Factor 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 Background OR Step 3 calculate Dilution Factor Press KHK to enter the Dilution Factor Screen Enter the Volume of the sample using the keypad numbers range 0 01 to 9999 Press the down arrow Enter the volume of the Diluent using the keypad numbers range 0 01 to 9999 Press OK to calculate the Dilution Factor and return to the Dilution Factor Parameters Screen Oligo Parameters OR Press to cancel the selections and return to the Parameters Screen Yolume Step 4 Diluent Select whether the Background Correction is to be used or not with the left and right arrows Press the down arrow Step 5 Select the Units of measurement using the left and right arrows Options ug ml ng ul ug l and pmol ul If pmol l is selected the factor changes to a selection table denoting the ratios of the 4 bases in the structure Press the down arrow Step 6 Units not pmol ul Enter the factor using the keypad numbers The default value is
75. o these as well as the final result on the right hand side Use of Background Correction Background Correction at a wavelength well apart from the nucleic acid or protein peaks is often used to compensate for the effects of background absorbance The procedure can adjust for the effects of turbidity stray particulates and high absorbance buffer solutions NanoVue uses Background Correction at 320nm by default on all Life Science Applications particularly nucleic acid measurements It is particularly recommended since very small samples are particularly susceptible to Stray particulates The Background function toggles On and Off with either left right arrows from the relevant page If 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 ratio 260 280 Abs ratio 260 230 Abs260 Abs320 Factor Abs260 Abs320 Abs280 Abs320 Abs260 Abs320 Abs230 Abs320 Version 4 0 Page 17 Typical spectral scan of a Nucleic Acid PURE NUCLEIC ACID POLY dAdT Wave 260_0 Abs 0_ 700 Wave 280 0 Abs 0 383 250 0 275 0 Wavelength Note e The absorbance maximum near 260nm and absorbance minimum near 230nm e The flat peak near 260nm and steep slope at 280nm e There is very little absorbance at 320nm Version 4 0 Page 18 4 2 2 DNA measurement The procedure is as follows DAWA Parameters Step 1 P
76. o to step 9 or New Standards means new standards are measured each time the method is used Step 8 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 2 or 3 Press Next to enter the Standards Screen OR Press Cancel to cancel selections and return to the Protein Screen Standards Screen Step 9 Standards Manual selected 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 backspaces and clears the last digit entered Step 10 Standards Manual selected Press Next to enter the Calibration Screen OR Press Back to return to the Parameters Screen Page 46 Biuret Calibration i fia O14 O86 OF LO Le L4 Biuret Calibration Standards 0 200 O 044 4 0 400 01484 T4 0 600 0 800 1 000 odara aa 1 400 0 249 4 Li 0 349 4 0 542 A Ot ie i tr DOE D04 ALE CE LO Le L4 Biuret Calibration Biuret Calibration Standards 0 700 0 400 0 145 A o 4 0 044 A 0 600 0 249 4 Li 0 800 1 000 o447a a 1 400 0 349 4 0 542 4 Ot F T tr fe D04 jo OE LO Le L4 mm Version 4 0 Calibration Screen Replicates off This shows the calibration values and allows standards to be measured or entered using
77. ons supplied Version 4 0 Page 85 LEGAL GE imagination at work and GE Monogram are trademarks of General Electric Company 2007 General Electric Company All rights reserved First published Nov 2007 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information http www gehealthcare com lifesciences GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences ABBj rkgaton 30 751 84 Uppsala Sweden GE Healthcare Europe GmbHMunzinger Strasse 5 D 79111Freiburg Germany GE Healthcare Bio Sciences Corp800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KKSanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan Version 4 0 Page 86
78. oving parts which contributes to its inherent reliability Figure 1 NanoVue Spectrophotometer General Principles In conventional UV Visible spectrophotometers the sample is usually contained within a glass or silica cuvette which is placed in the sample beam The absorbance of this is measured and then compared to that of a standard From this comparison the concentration is calculated However when the quantity of sample is limited or highly concentrated dilution or the use of ultra low volume cuvettes is required which is time consuming can introduce errors and presents cleaning difficulties NanoVue was developed to overcome these problems Using NanoVue typical sample volumes of 2ul are pipetted onto a hydrophobic surface and then a very short pathlength of either 0 2mm or 0 5mm is created by lowering the sampling head onto the top of the sample With the sample so constrained all of the general software features of the instrument including wavelength scanning single or multi wavelength absorbance and concentration measurement kinetics standard curves and absorbance ratio can be utilized In all of these modes the instrument utilizes either the 0 2mm or 0 5mm pathlengths The benefit of these small pathlengths is that the instrument can measure smaller volumes of very concentrated or highly absorbing samples NanoVue also has a range of more specific life science applications including DNA and RNA concentration and pur
79. ow keys to go to the Lid Switch option and select On Theme Lid Switch to turn Auto Read on or Off to turn Auto Read off Press the confirm button ee When Auto Read is off use the 0A 100 T and keys to take the reference and measurement scans respectively When Auto Read is on the keys on the keypad are still E functional This enables a user to carry out a new reference scan at any time by pressing the 0A 100 T key prior to lowering the sampling head or to perform a series of readings or reference scans without raising the sampling head 2 7 Quality assurance Quality Assurance is provided to help minimize the impact of pipetting errors If Quality Assurance is switched on from the Preferences Screen the instrument will ask the user for a second reference to compare with the first to ensure that the reading is reasonable If the second reading is not sufficiently close to the first reading the user will be asked to replace the reference again The process will be repeated until two successive good readings within 0 02A are identified and the reference will be the average of these readings Note When the user is prompted for the second reference the user is required to clean the surface and reapply the sample The 0A 100 T key is disabled until the user lifts the sampling head to prevent the user from just re reading the same sample 2 8 Keypad and display The back lit liquid crystal display is very easy to navigate around using
80. ple plates are clean have been fitted securely and are well seated Is the xenon source firing when you press 1 Can you hear it buzz at start up Is the power supply securely connected to the instrument Turn off the instrument disconnect and reconnect the power supply and then turn on the instrument If the problem persists please contact your supplier If any calibration error messages are seen do not use the instrument for measurement Check the instrument as indicated and contact your local supplier should the problem persist Version 4 0 Page 82 9 3 Fault analysis Instrument fails start up calibration Instrument switches off after calibration Instrument switches off during measurement No image on screen Version 4 0 Check that you are using the correct power supply It should be an 18V unit Ensure that the connector is pushed in fully at the back of the instrument If the problem persists please contact your supplier You may be keeping you finger on the ON OFF button too long so that the instrument receives both ON and OFF signals and switches off after the calibration Try adjusting the timing of your finger press at switch on lf the problem persists please contact your supplier Check that you are using the correct power supply It should be an 18V unit Ensure that the connector is pushed in fully at the back of the instrument lf the problem persists please contact your supplier If the lamp c
81. ppropriate factor should be used to convert them x20 for 0 5mm pathlength x50 for 0 2mm The procedure is as follows Step 1 Wavescan Parameters Set Start Wavelength by using keypad numbers or left and right arrows range 200 940nm Start wavelength Pathlength Press the down arrow Key Step 2 Set End Wavelength by using keypad numbers or left and right Poe oneienain arrows range 210 950nm eee Press the down arrow key Step 3 uia Select the Mode Absorbance or T using the left and right Arrows Step 4 oo Select Pathlength from 0 5mm or 0 2mm Step 5 To enter the Measurements Screen with the selected parameters press OK OR Wavescan Cancel the selections and return to the Applications Screen by pressing Cancel E Step 6 Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 7 Clean the top and bottom plates pipette on the sample and lower the sampling head If Auto Read is off press FE Repeat step 7 for all samples Results A graph of the Wavescan is displayed along with a table of Absorbance T at each peak Use the left and right arrows to move the cursor along the graph When it reaches a peak the peak height and width of the peak is displayed at the top of the screen To zoom in on the
82. ress 1 to select DNA mode Pathlength Units Step 2 Select Pathlength using the left and right arrows Options are 0 5mm 0 2mm or Automatic Press the down arrow Dilution Factor Factor Step 3 Dilution Factor known ao Enter the Dilution Factor using the keypad numbers range 1 00 to 9999 Use the C button to backspace and clear the last digit Background entered On OR Step 3 calculate Dilution Factor eo Cancel Press KIM to enter the Dilution Factor Screen see second parameter screen to the left Enter the Volume of the sample using the keypad numbers range 0 01 to 9999 Press the down arrow Enter the volume of the Diluent using the keypad numbers range 0 01 to 9999 TMA AmA Press OK to calculate the Dilution Factor and return to the Parameters Screen OR Press Cancel to cancel the selections and return to the Dilution Factor VE Parameters Screen Step 4 Background Correction at 320nm will be on It may be turned off Diluent with the left or right arrows Press the down arrow Step5 Select the Units of measurement using the left and right arrows Options ug ml ng ul ug ul Press the down arrow Step 6 i Enter the Factor using the keypad numbers The default value is 50 the range is 0 01 to 9999 Step 7 Press OK to enter the Results Screen and begin making measurements OR to return to the Life Science Screen Results Screen Step 8 Pipette on the re
83. result calculated from the selected parameter dA final A or slope 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 Press Cancel to return to the Applications Screen Press KHK to display available Options which are described below Version 4 0 Page 58 Parameters Print Frint O ata Set tO At Cursor Set tn At Cursor Slope Sauve biethod Subo Frint a 3 G O 8 Version 4 0 Sample MHumber Options select using keypad numbers 1 2 3 4 Exit Options by pressing Return to Parameter 1 Screen step 1 above Print data on the results screen via selected method Print all the data Set the t position starting point for the slope and dA calculation at the current cursor position Value is retained for subsequent samples Set the t position finishing point for the slope and dA calculation at the current cursor position Value is retained for subsequent samples Toggle the calculated slope line on and off Note if any data points enclosed by t and t are beyond the range of the instrument gt 2 5A or lt 0 3A then this option is greyed out Sample Number add a prefix to the sample number and reset the incrementing number to the desired value Save Method use the left and right arrows to select a folder to store in Favourites Methods
84. retical absorbance factor respectively of an entered base sequence Tm This is calculated using the equation i AH x 100 273 15 16 6 log salt AS 1 987 x log c 4 53 0822 where AH and AS are the enthalpy and entropy values summed from respective 2x4x4 nearest neighbor matrices c is the Primer concentration of oligonucleotide pmoles ul in the calculated Tm or the measured concentration in measured Tm In the latter case concentration is obtained from the equation c Abs 260nm x Calculated factor x pathlength multiplier x 10 000 MW Calculated factor and MW are defined below salt is the buffer molarity plus total molarity of salts in the hybridization solution moles I Weights for AS are indexed by adjacent paired bases A similar equation applies to weights for AH again indexed by adjacent bases Note that bivalent salts may need normalizing using a multiplying factor of 100 because of their greater binding power Theoretical Absorbance The Theoretical Absorbance is based on a calculation as follows For each adjacent pair of bases nearest neighbors an extinction coefficient weight is accumulated using a 4x4 table one for either DNA or RNA This total weight is doubled and then for each internal base a counterweight is subtracted using another 1x4 table The end bases are excluded from the latter summation That is Total Extinction Coefficient E x 2 x alable base_type base n base n 1 tTable b
85. rom both the top and A bottom sample plates Apply a drop of the calibration fluid by gently 2 2 2 2 A JA m Aa a E m A ma ae Eoi Ea E mm G squeezing the bottle Press FW to measure the absorbance of the calibration fluid again it will take longer than normal Repeat this process until the table of results is full Results which are obviously poor can be overwritten by moving the highlight bar up to the poor result pressing C to clear the line and re reading the calibration fluid Very bad readings gt 0 15A background absorbance or very low or high readings will be rejected by the instrument When the table is full press OK i anda pop up will appear to inform the user if the pathlengths have been updated successfully or not Press OK again and the Results Screen will be shown Results Screen The Results Screen shows the measured absorbance at each of the two pathlengths These should be close to 0 5 for the 0 5mm pathlength and 0 2 for the 0 2mm pathlength the allowed ranges are 0 45 0 55 and 0 18 0 24 respectively The actual values measured are stored by the instrument and used subsequently to compensate for the inaccuracies in the two pathlengths meaning the results are always presented as though the pathlengths were perfect Version 4 0 Page 71 Error Message In exceptional circumstances the measurements will not be close enough to 0 2 and 0 5 and an error message is generated
86. rs Seinen j Options mg ml ug ml ng l and yg ul Step 6 Mode Molar extinction Beihai ap a aa t Enter the Value for the molar extinction coefficient of the protein reference being used units I mol The default value is 50 Press the down arrow Step 7 Mode Molar extinction Enter the Molecular Weight of the reference protein in kilo Daltons The default value is 50 eK Step 6 Mode Mass extinction Enter the Mass Extinction Coefficient for the protein reference being used units I g The default value is 50 Step 6 Mode E 1 Enter the Mass Extinction Coefficient for a 10 mg ml 1 solution of the reference protein The default value is 50 Step 8 all modes Dilution Factor 1 000 Press OK to enter the Results Screen OR Cancel to return to the Protein Screen Version 4 0 Page 35 Results Screen Protein 4780 Step 9 A260 23A Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used A320 0172A for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key 0 1 88 pressed again Step 10 Clean the top and bottom plates pipette on the sample and lower the sampling head If Auto Read is off press T This measures at both 260 and 280nm wavelengths and displays the Protein concentration as the Result Repeat step 10 for all samples Press to retur
87. s Screen Step 8 Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for RNA all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 9 Clean the top and bottom plates pipette on the sample and lower the sampling head If Auto Read is off press MWL This measures at the selected wavelengths and displays the results a0 The ratio of the absorbance values at wavelengths 1 and 2 are calculated The Concentration is based on the absorbance at DRNA wavelength 1 e Repeat step 9 for all samples Press to return to the Life Science Screen Press KKM to display available Options which are described below Version 4 0 Page 21 OO tolon Version 4 0 Parameters F rint Graph Sample Mumber Save Method Swubo Frint Exit Options by pressing Options select using keypad numbers Return to Parameters Screen step 1 above Print result via selected method Toggle Graph on off The graph shows a Wavescan plot across the range 220nm to 320nm with cursors denoting 230 260 280 and if Background Correction selected 320nm Sample Number add a prefix to the sample number and reset the incrementing number to the desired value Save Method use the alpha numeric keys to enter a name for the method and press Save Auto Print toggles Auto Pri
88. s Standard Curse Multi wavelength Ping o Absorbance A atio SUMMARY Function Keypad Number Description O 1 Absorbance or T transmission at a single user defined Single wavelength wavelength 2 Concentration measurement at a single wavelength based on a factor entered or calculated from a single standard Concentration 3 Wavelength scan between two user defined wavelengths Range 200 900nm with user configurable peak finding function Wi aVescan 4 Absorbance versus time measurements either rate or end value Kinetics based 5 Generation of calibration curve by measuring standards at a single Standard Curve wavelength G 6 Absorbance or T transmission at up to 5 user defined Muli wavelength wavelengths o Pina 7 Ratio of absorbance values at two user defined wavelengths Absorbance A atio OPTIONS Within each application the user has the possibility of selecting various options that define the way results are treated If not using a stored method it is advisable to check that these options have been appropriately set for your experiment when coming to the instrument Note that setting the History parameter to on see Preferences later will cause the instrument to store its last settings If the History parameter is turned off all parameters and options will return to their default settings when you leave that application unless it has been saved as a method Version 4 0 Page 49
89. sample will need to be replaced and the 0A 100 T key pressed again Step 12 Standards selected Press replicates to display the replicate entry boxes Use C to clear previously stored results before measuring Clean the top and bottom plates pipette on the replicate standard and lower the sampling head If Auto Read is off press AE to measure the standard and store the result Press Next to move from replicates of one standard to replicates of the next standard A graph will display the results and the fitted curve as the measurements are input Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 Standards Manual selected Press Next to accept the calibration and go to the Results Screen see below OR Press Back to return to the Standards Screen Page 44 Calibration Manual entry Lowry Calibration Shows previously entered calibration values and allows values to be entered via the keypad l The highlighted box can be edited in order to enter an are y absorbance value corresponding to a given concentration value using the keypad numbers range 0 001 to 9999 Use C to 3 backspace and clear the last digit entered and the up and down moai arrows to move between boxes 0 050 A coro mu 0 030 A 0 110 01304 oan 0 150 4 me Press OK to accept the calibration and go to the Results TE LH O8
90. sampling head is lowered into position and the reading taken The pathlength is automatically adjusted unless the user selects manual adjustment This can be either 0 2mm or 0 5mm Application of the sample is most conveniently carried out using any commercially available pipetting tool and standard tips a 10ul pipette is recommended Figure 3a Sample head Figure 3b Sample application 2 5 Pathlength and absorbance normalisation For methods in the Application folder the absorbance values have not been normalized and the pathlength needs to be taken into account when the values are used for concentration calculations NanoVue has the ability to measure samples using either a 0 2mm or a 0 5mm pathlength The 0 5mm pathlength should be used for samples of low concentration and the 0 2mm pathlength for more concentrated samples It is best to use the 0 5mm pathlength whenever possible In the Life Science applications the absorbance readings are presented as their nomalized 10mm pathlength values to allow the use of literature based factors for concentration measuremenis Within the DNA RNA Oligonucleotide Tm Calculation Cy Dye Protein UV and Protein A280 methods the system defaults to having the pathlength chosen automatically When this is active the instrument will measure at the 0 5mm pathlength first and if the absorbance is high gt 1 7A then the measurement will be carried out at the 0 2mm pathlength If the concentration range of
91. ss Back to return to the Standards Screen Results Screen Step 14 Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 15 Clean the top and bottom plates pipette on the sample and lower the sampling head If Auto Read is off press FEL The concentration of the sample is taken and displayed Repeat step 15 for all samples Press to return to the Protein Screen Press KRM to display available Options which are described below Options select using keypad numbers 1 Return to Parameters Screen step 1 above 2 Print result via selected method 3 Toggle Graph on off Displays the calibration graph cursors give values for last measured sample 7 Sample Number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save Method use the alpha numeric keys to enter a name for the method and press Save 9 Auto Print toggles Auto Print on off Esco Exit Options by pressing Or wait Page 42 4 3 6 Lowry The procedure is as follows Step 1 Lowry Parameters Press 5 to select Lowry method Step 2 Pathiength The Wavelength for this method is fixed at 750nm Step 3 Enter the number of Standards 1 9 to be used in the curve using the keypad
92. t Page 28 4 2 6 Cy Dye measurement Measurement of the labeling efficiency of fluorescently labeled DNA probes before 2 color microarray hybridization ensures that there is sufficient amount of each probe to give satisfactory hybridization signals These data also provides an opportunity to balance the relative intensities of each fluorescent dye by adjusting the concentration of each probe before hybridization The DNA yield is measured at 260nm while the incorporation of fluorescein Cy3 and Cy5 are measured at their absorption peaks This method may also be useful for measuring the yields and brightness of fluorescently labeled in situ hybridization probes Cy Dye Parameters Step 1 Press 5 to enter the Cy Dye Parameter Screen Enter the Dye Name of the dye being used the default is Cy3 but others may be generated using the Clear key C and the alphanumeric keypad Press the down arrow Step 3 Serene Enter the Wavelength of the dye peak absorbance Press the down arrow Step 4 Enter the dye Coefficient of extinction Press the down arrow Step 5 Enter the Pathlength of the sample cell Options are 0 5mm 0 2mm or Automatic Press the down arrow Dilution Factor Start RNA Step 6 a Select whether Background Correction is on or off Cy Dye Settings Step 7 Press Next to enter the Settings Screen oo OR Volume Press Cancel to return to the Life Science Screen EE Step 8 Dilution Factor known Enter the Dilutio
93. t Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 Standards Manual selected Press OK to accept the calibration and go to the Results Screen see below OR Press Back to return to the Standards Screen Page 38 BCA Calibration Calibration Manual entry Standards Shows previously entered calibration values and allows values to 0 200 oosa 3 i be entered via the keypad 0 400 oina me tf The highlighted box can be edited in order to enter an 0 600 0 288 4 e eee absorbance value corresponding to a given concentration value 0 800 0 404 A iA using the keypad numbers range 0 001 to 9999 Use C to 1 000 asea o backspace and clear the last digit entered and the up and down 1 400 ez BE arrows to move between boxes Pressing the down arrow from az the last standard will bring up the OK box na 0 4 16 O18 lO lE 4 Press OK to accept the calibration and go to the Results Screen see below OR Press Back to return to the Standards Screen BCA Results Screen Step 14 Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for wavelength aes all subsequent samples until changed If QA is switched on the WAGE sample will need to be replaced and the 0A 100 T key pressed again Curve Fit Step 15 EE Clean the top and bottom plates pipette
94. ters 1 View Parameters for the experiment E Frin 2 Print the results Frin ata 3 4 5 6 Described in the relevant application CP Sett0 At Cursor 7 Define the Sample Number you wish to start from GP Setin At Cursor 8 Save the parameters as a method to a defined folder Slope name with a defined method name D CENS 9 Toggle Auto Print on off Default is off Save Method J C Auto Print vi Exit options by pressing Esc or wait Experienced operators can use the numeric keys as a shortcut to the option required without needing to enter the Options menu 2 9 Software style The user interface is built around folders of files which are displayed on the first page when the instrument is switched on Different folders are numbered and opened by using the associated number key on the keypad o9 Life Science eQ Applications eQ fl Favourites oQ Methods Utilities Summary Function oQ Life Science e9 Applications o R Favourites oQ hl ethos eQ Utilities Version 4 0 Keypad Number Description Standard life science applications such as nucleic acid and protein assays General spectroscopic applications A folder to store your more frequently used methods Inactive when empty Contains 9 folders that can store less frequently used methods Up to 9 methods per folder are allowed totaling 81 methods Instrument set up options and gam
95. th and divide by 5 for the target value for the 0 2mm pathlength Press OK to carry on with the Calibration or Cancel E to return to the Utilities Screen Step 3 Open the NanoVue sampling head and pipette 5ul of distilled water onto the correct position on the sample plate To reduce small errors being introduced due to droplet misplacement the higher volume is recommended Close the sampling head clear all previous readings using the cursors and C key Press the OA 100 T key as in section 7 7 This will need to be done twice to complete reference QA Step 4 Figure 22 Open the sampling head clean off the water from both the top and bottom sample plates Apply a drop of the calibration fluid by gently squeezing the bottle Press c to measure the absorbance of the calibration fluid Observe the 0 5mm value and compare to the calculated value for the 0 5mm pathlength found in Step 2 If larger adjust the left hand screw anti clockwise about a turn as shown in figure 22 if smaller turn clockwise To adjust the 0 2mm pathlength value turn the right hand screw clockwise to increase the value anti clockwise to reduce see figure 23 Once the adjustment is made open the head and replace the calibration droplet Press MEL to see the effects of the adjustments Repeat this process until the value is within 0 02A of the target value for the 0 5mm pathlength and 0 015A for the 0 2mm pathlength Step 5 Results can
96. the calculated molecular weight divided by the theoretical absorbance Version 4 0 Page 26 The procedure is as follows Tm Calculation Parameters Step 1 Press 4 to enter the Tm Calculation Parameters Screen Base Type Buffer Molarity Step 2 Select the Base Type DNA or RNA Press the down arrow Select whether the sample is Phosphorylated or not Yes or No Press the down arrow Primer Conc Step 4 Enter the Primer Conc using the keypad numbers range 0 000 to 99 9 in pmole ul Press the down arrow Step 5 Enter the Buffer Molarity buffer molarity total molarity of salt in Tm Calculation Parameters moles l using the keypad numbers range 0 000 to 10 Press the down arrow Base Type Buffer Molarity Step 6 Select the Counter lon Na K TEA or Other Step 7 if Other selected see the previous section Phosphorylated Counter lon Enter the molecular weight Other MW of the Counter lon used Step 8 a SF eae re Press Next to select these parameters and go on to the Press Cancel to return to the Life Science Screen Tm Calculation Base Sequence Step 9 RES Select the Pathlength of the sample cells Options are 0 5mm 0 2mm or Automatic Base Sequence Tm Calculation Base Sequence Step 10 Enter the known Base Sequence triplets using the number keys 1 for A 3 for C 4 for G and 6 for T or U Note that a comma is added after each triplet to improve readability Pathlength Base Sequence ACG
97. the keypad numbers if calibration mode is manual Step 11 Standards selected Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 12 Standards selected Clean the top and bottom plates pipette on the standard and lower the sampling head If Auto Read is off press MWL use C to clear previously stored results before measuring to measure the standard and store the result Repeat step 12 for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 Standards Manual selected When all standards are measured entered the OK box appears Press OK 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 Step 11 Standards selected Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be r
98. the samples is known beforehand the relevant pathlength can be pre selected if it is unknown then the automatic option can be used Within the other applications the actual measured absorbance value is reported so if the absorbance is high at the 0 5mm pathlength gt 1 7A the sample can be re measured at the 0 2mm pathlength In the automatic mode the reference scan is always carried out using both pathlengths so that if a sample is measured at one pathlength and subsequently re measured at the other pathlength a new reference scan is not required The pathlength that is used to carry out the measurement is printed exported with each result and is also displayed in the top left hand corner of the header line on the instrument s display Care must be taken that the correct pathlength is being used when comparing sample results or using a stored calibration curve 2 6 Auto Read NanoVue senses when the sampling head has been lowered so Auto Read can be used to automatically make a measurement when this happens obviating the need to press any keys The instrument assumes that the first reading after starting any application will be a reference scan and that subsequent readings will be sample scans Version 4 0 Page 8 Single wavelength 0 5mm pathlength 0 2mm pathlength Preferences Switching Auto Read on or off Sense Auto Standby Go to Utilities option 5 from the NanoVue menu a ee Select Preferences option 4 Use the arr
99. tion to the protein concentration due to absorbance at 260nm e Set Factor 2 0 00 for direct A280 UV protein measurement Factor 1 is based on the extinction coefficient of the protein If BSA bovine serum albumin is an acceptable standard setting Factor 1 1 493 will give linear results from 0 1 to 50 mg ml protein Protein mg ml 1 493 Abs280 e Inthe Protein A280 application various modes can be selected depending on the extinction coefficient of the reference protein being used A If the mode is Christian Warburg the equation given above is used B For BSA the extinction coefficient is 6 7AU E1 at Abs 280 for a 1 ww solution C For IgG the extinction coefficient is 13 7AU E1 at Abs 280 for a 1 ww solution D For lysozyme the extinction coefficient is 26 4AU E1 at Abs 280 for a 1 ww solution E For other reference proteins the user can enter the molar extinction coefficient and the molecular weight of the protein the mass extinction coefficient for a 1 solution or the E1 value The expressions are Molar Extinction Conc in ug ml A280 kDa 1000 em where em molar extinction coefficient em M cm kDa molecular weight in kiloDaltons Mass extinction Conc in ug ml A280 1000 ema where ema mass extinction coefficient ema equivalent to absorbance of 1 g l solution E1 Conc in ug ml A280 10000 E1 where E1 extinction coefficient E1 g 100ml cm equivalent to the
100. umol 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 0 to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters or Cancel E Step 5 Enter the Pathlength Options are 0 5mm or 0 2mm Press Next to move on to the next Parameters Screen or Es Cancel to return to the Protein Screen Step 6 Enter the type of curve fit Options are Regression straight line Zero Regression forces the straight line through the Bradford Parameters origin Interpolated or Cubic Spline Press the down arrow Curve Fit Step 7 slate asia Select the calibration mode either Standards measure prepared standards Manual keypad data entry go to step 9 or New Aia Standards means new standards are measured each time the Pe method is used Step 8 only 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 2 or 3 Press Next to enter the Standards Screen Replicates OR Press Cancel to cancel selections and return to the Protein Screen Standards Screen Step 9 Standar
101. urement The procedure is as follows Step 1 HMAC Parcmsics Press 2 to select RNA mode Step 2 Pathlensth TE Select Pathlength using the left and right arrows Options are 0 5mm 0 2mm or Automatic Press the down arrow Step 3 Dilution Factor known MRON FAAI Factor 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 Background OR Step 3 calculate Dilution Factor Press XII to enter the Dilution Factor Screen see second image to the left Enter the Volume of the sample using the keypad numbers range 0 01 to 9999 Press the down arrow Enter the volume of the Diluent using the keypad numbers range 0 01 to 9999 Press OK to calculate the Dilution Factor and return to the Parameters Screen OR Press to cancel the selections and return to the Parameters Screen Dilution Factor Step 4 Select whether the Background Correction at 320nm is to be Bolume used or not with the left and right arrows l Press the down arrow Step 5 mateak Select the Units of measurement using the left and right arrows Options ug ml ng ul ug yl Press the down arrow Cancel Cancel Step 6 Enter the Factor using the keypad numbers The default value is 40 the range is 0 01 to 9999 Step 7 Press OK to enter the Results Screen and start making measurements RHA Parameters me OR to return to the Life Science Screen Result
102. urement using the left and right arrows Options ug ml ng ul and yg ul Step 8 Press OK to enter the Results Screen OR Cancel to return to the Protein Screen Version 4 0 Page 33 OO DoS Version 4 0 Protein UW Parameters F rint Graph Sample Mumber Save Method Subo Frint Results Screen Step 9 Pipette on the reference sample and lower the sampling head If Auto Read is off press the 0A 100 T key This will be used for all subsequent samples until changed If QA is switched on the sample will need to be replaced and the 0A 100 T key pressed again Step 10 Clean the top and bottom plates pipette on the sample and lower the sampling head If Auto Read is off press EWL This measures at both 260 and 280nm wavelengths and displays the Protein concentration as the Result Repeat step 10 for all samples Press to return to the Protein Screen Press KHK to display available Options which are described below Options select using keypad numbers 1 Return to Parameters Screen step 1 above 2 Print result via selected method 3 Toggle Graph on off The graph shows a wavescan plot across the range 220nm to 330nm with cursors denoting 230 260 280 and if background correction selected 320nm 7 Sample Number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save Method use the alpha numeric keys to enter a name
103. urs on a steep slope where small changes in wavelength will result in large changes in absorbance Consequently small variations in wavelength accuracy have a much larger effect at 280nm than at 260nm It follows therefore that the 260 280 ratio is susceptible to this effect and users are warned that spectrophotometers of different designs may give slightly different ratios In practice concentration also affects the 260 280 ratio as the individual readings approach the instrument s detection limit If a solution is too dilute the 280nm reading shows a greater proportional interference from background and as the divisor becomes smaller has a disproportionate effect on the final result It is advisable to ensure that the Abs260 value is greater than 0 1A for accurate measurements An elevated absorbance at 230nm can also indicate the presence of impurities 230nm is near the absorbance maximum of peptide bonds and may also indicate interference from common buffers such as Tris and EDTA 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 which absorbs over the 230 260nm range A wavelength scan of the nucleic acid is particularly useful for RNA samples NanoVue displays the individual absorbance values and the 260 280 and 260 230 ratios on the left side of the screen and attention should be given t
104. wavelength scale use the up arrow This auto scales on the Absorbance T scale dependent on the Graph Scale option and this is retained for subsequent measurements To zoom out again use the down arrow a avescan Press to return to the Applications Screen Press KHK to display available Options which are described next Version 4 0 Page 54 Min Pk Height Sort Peaks By Min Pk idth Draw Peaks Parameters Print Abs 38T Peak Detection Add Peak Graph Scale Sample Mumber Save Method Auto Frint L Ei Q O 6 Peak Detection Auto detect Peaks Peak Detect on Zoom coe Sample 1 Version 4 0 a avescan Height 0 0354 width 12 0n0mi Options select using keypad numbers 1 Return to Parameters Screen step 1 above 2 Print result via selected method 3 Toggle between Absorbance and T mode 4 Displays Peak Detection Parameter Screen See description below Manually adds a peak position to the peak table in the results screen at the position set by the cursor If the cursor is returned to this position the legend User Defined Peak is displayed at the top of the scan and this option changes to Delete Peak 6 Displays Graph Scale Parameter Screen See description below 7 Sample Number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save Method use the left and right arrows to select
105. when off the keypad must be used to take readings 6 Quality Assurance function can be on or off Press to store the settings and return to the Utilities Screen or press to return to the Utilities Screen without storing the settings 7 5 Contrast Ambient temperature can affect the display This function can optimise the display for local conditions The procedure is as follows Contrast Adjust the Contrast using the left and right arrows to scroll through the levels Press the down arrow briefly to move down Adjust the brightness similarly Brightness Press the down arrow Press OK to store the settings and return to the Utilities Screen Contrast 7 6 About Displays the instrument serial number and software version GD Serial Number q rrrz15 Yersion 42752 41 0 Press OK to close the window and return to the Utilities Screen Version 4 0 Page 70 7 7 Pathlength Calibration lt is recommended that the pathlength is calibrated either at a frequency determined by the standard operating procedure of the user s laboratory at a minimum of once every six months or when the sample plates are changed Step 1 Papen deo aen a amerers Enter the certificate ID on the certificate bottle of the calibration solution using the keypad Press the down arrow as Step 2 Enter the calibration value of the calibration solution Calibration value Press OK to carry on with the Calibratio
106. y and should not be covered up Switch on the instrument via the keypad after it has been plugged in The instrument will perform a series of self diagnostic checks It is recommended that users read through this manual prior to use Contact your supplier if you experience any difficulties with this instrument 1 2 Safety Caution Strong light source Never look directly into the beam of any UV Visible spectrophotometer The instrument is fitted with two safety interlocks The first prevents operation with the sampling head raised the second limits operation to a single xenon source flash if no light reaches the detector If the instrument is used in a manner not specified or in environmental conditions not appropriate for safe operation the protection provided may be impaired and instrument warranty withdrawn There are no user serviceable parts inside this instrument Version 4 0 Page 4 1 3 Declaration of conformity GE Healthcare certifies that NanoVue UV Visible Spectrophotometers part numbers 28923215 28923216 28923217 28924402 289403 28924404 conform to the requirements of the following Directives 73 23 EEC and 89 336 EEC and IVD Standards to which conformity is declared EN 61010 1 2001 EN 61326 2 3 1998 EN 61000 4 6 1992 2002 96 EC RoHS Statement EU China Version 4 0 Safety requirements for electrical equipment for measurement control and laboratory use Electromagn

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