µl Spectrophotometer USER MANUAL Version 4.0/21/03/11 All
Contents
1. Absorbance Wavelength il rps emm TI 000005 irgi 900005 E Irgl con A D egal E Ivuirsp ot Specronater Read Petia made Porter O Example Zoom using Pointer selected Multiple overlays can be easily differentiated Alternatively Cursor can be chosen from the drop down menu at the bottom of the graph window When Cursor is chosen the mouse will move along the graph only and display the relevant absorbance and wavelength to the right of the graph GH Ainon o E 4 Kn d Pointer Om E sorbance Wavelength YA Pxodeop i Spexooneter Ready Pipette mode Cursor Cle Crenrtays Peng Zoom Example using Cursor cross hairs Printing Results can be printed by any printer configured for use on the host PC this includes PDF and other document converters that can be viewed as printers too A single result can be printed complete with the scan and set up data appended or multiple results can be selected and printed in a tabulated format In addition an optional label printer is available for printing directly on to sticky labels that can then be stuck in log books or on sample containers The label size is 62 x 100mm Printer Label Layout not to scale Printing of individual results together with the graph and measurement details can be achieved from the Project Selector page Choose the project in which your results of interest have been saved2. Setting the IP Addresses in a Windows Vista Systems amp Windows 7 Open the Control Panel by pressing the Start button and then selecting Control Panel H 7 TERLER 2 xd A mai EO A oe z SG 6 de S vs mm E mec om mee 5 P DW r A RE a E md ost huest kat deg deng e OG oO o sg 0 E wa E H amp gd La sg B e e d SC we SE ES o we I KS SS gy WW vi Si ug m We Now select Network and Sharing then select manage network connections from the menu on the left you may need to consult the manual for your computer system to find out which controller to use EC rete r H lt D e wm R mm mp Aa af LG AAA a reg AA 9e r i o E Facies Network ssd Qua Certes IL D d ws T pee mr rmt rege or een rr A L Zei inr far catus saa set el Ma rear teg tmt md ctm aias Prosa ramet Sue _ tv Commu voan Fut eoi Lowe een Tage wa rats 4d Sqna ew Tankas Duae t Matron Sacco om LN mm ers em si Publ bam geng d Trend om pasear pam si Der reeg ear si famem vcn m ann em si Veda geg CEA Zen re di fu fen eegent er ged et ia rote 99 591 md lem Zen we d fu fuc ra fees a fu emt tree DX m fermes rr Double click on the wired Ethernet connection shown in the Local Area Connection V Sy ER A SEA R eege NA mea A i avi
3. requesting that you re blank will be displayed If this occurs simply try blanking again and if the procure continues to fail try following the lens cleaning protocol The Microarray mode is designed to report the ABSORBANCE DYE CONCENTRATION pmol ul and RATIO DYE BASE at 2 chosen wavelengths defined by the 2 dyes you have chosen to measure To choose your dyes go to Select Dye shown at the bottom of the screen Clicking on the tab will display a list of preset dyes to choose from for both dye 1 and dye 2 Additional dyes can be added by clicking on Editor NOTE WAVELENGTHS MUST BE CHOSEN BEFORE PRESSING MEASURE otherwise no data will be reported Additional information displayed in this mode includes Nucleic acid concentration ng ul Nucleic Acid purity 260 280 ratio Concentration of each dye C Absorbance for each dye A Ratio of dye base R paso IM m e Pxodrup Microarray pmol uL S 4 Eeer ed MO OAL 01082000 1201 WC vo 117647 on 15710432 DH OAC 03 02 2008 1204 DUNC v 118 842 1587 0 2 7 247 D2L 02 02 2009 12404 DBC YS v 658 558 325 257 555 EH 03 02 2008 12 06 Calculating the Labelling Efficiency and Concentration of Nucleic Acid The relative efficiency of a labelling reaction can be evaluated by calculating the approximate ratio of bases to dye molecules This ratio can be determined as described below by measuring the absorbance of the nucleic acid at 260 nm an
4. Reference Only The spectrometer has the IP address of 192 168 1 200 Your wired Ethernet parameters are as follows IP address 192 168 1 nnn any number from 2 254 except 200 is valid Subnet mask 255 255 255 0 Default gateway 192 168 1 1 DNS 192 168 1 1 If you experience any issues with connection please contact your local Picodrop representative who will be able to assist Instrument Start Up Once the installation has been completed successfully double clicking the desktop icon will allow login as a guest or administrator IMPORTANT IT IS IMPORTANT TO ALLOW THE UNIT TO WARM UP FOR 10 MINUTES BEFORE USE THIS WARM UP TIME ALLOWS THE LAMP TO STABILISE AND ENSURES CONSISTENT LIGHT INTENSITY Logging in to the Software On starting the software you will be shown a Login box The default login username is guest and the password is left blank Simply clicking OK will allow entry to the software as a standard user Isername If multiple users will be operating the instrument then the system administrator should set up accounts for each user To login as system administrator use the following username admin password password The Administrator can reset the above password and create edit delete other standard user accounts In the Administration page the user can choose Settings from the menu and change the tolerance on blanking not recommended to change and also the default number of m
5. and calculate the equivalent 10mm absorbance value It is the calculated 10mm absorbance value that is displayed on the Y Axis and in table and reports D Picodrop Application guest Project Selector E Existing Projects Sample Mame Date andTime Result test thick fibres thin fibres unit28 thin fibres unit 1U thick fibres unit 10 picocal unit 110 thick fibre picacal unit28 thin fibres picacal unit28 thick fibres picacal uniti 10 thick Fibre picacal warm unit28 thick DNA test of unit28 DNA test of uniti 10 DNA best of unite tip rotation test unit DNA 00 Cosmo JA final test India Testing m UE m zu Du B Opening an existing sample result and graph The project Selector window can be accessed at any time and by simply clicking on a project folder the user can view a list of sample results By clicking on any sample line the original graph and detailed data report will be displayed Picodrop Application guest Project Selector test thick fibres Sample Sample Mame Date and Time Result L i E 5 thin fibres unit28 thin fibres uniti 10 thick fibres uniti 10 picocal unit 110 thick f picacal unit28 thin fibre picacal unit28 thick fib picacal uniti 10 thick Fi picacal warm unit28 th DNA test of unites DNA test of unit 10 DIMA test of unite lip rotation test unith DNA100 Cosmo JH final test India Testing DNA 50 Meagsuren
6. and click on the line to highlight the specific test s you need to print Then simply press the PRINT button shown in the menu on the right side of the screen Multiple results can be selected using shift and click methods to enable printing of a tabulated version of the results without the graph Window ID Sample Name Date and Time Fendt 1 Open the project that contains the result for printing 2 Click the result s in this area to highlight them for printing 3 Click on Print button to open the Print Dialoque The Printing Control Panel is then opened allowing the selection of paper or label printing labels can only be printed subject to connection of the optional label printer From here it is also possible to see a Print Preview on the screen for verification before printing to save wasted paper it is good practice to preview your printout whenever settings have been changed or a new printer installed selected Printing Control Panel x After selecting the relevant print function Paper or Label the Printer Selector screen is opened to allow selection of the appropriate printer This will show all printers installed on the host PC and the currently connected printer should be selected for paper printing If the optional Label printer is connected and installed the Brother QL 570 option should be selected Select Printer Name Documents Status Comments Se Add
7. bow N Cn i 1 file e wa c i a 4 e Ee P pe G I 0 2 JA S Wa 7 1 3 14 3 he x a Netiireme G You will then usually be asked to confirm a restricted system action by the Windows UAC User Access Control then Select Continue to allow configuration of the network adapter NOTE It may be necessary to disable other networks if there is an IP address conflict this can easily be done in the screen above by right clicking on each network in turn and selecting the Disable option Any disabled network can be switched back on by carrying out the same procedure and selecting the Enable option Liver Account Contrel EI H you started this action continue KZ Network Cennectons Microsoft Windows wc Detiis User Account Control helps stop unauthorged changes to your computer Select Internet Protocol Version 4 TCP IPv4 ensuring that the checkbox to the left of the item remains checked Now click on Properties H Local Area Connection Properties E Client for Microsoft Networks dl Microsoft Network Monitor 3 Driver dall Virtual Machine Network Services MI E QoS Packet Scheduler v vi Re and deieren Networks Enter the values as below and click OK to save changes Make sure that there are no other network adapters active you may need to consult your system s user manual to achieve this Internet Protocol Version 4 TCP IPv4 Properties Technical Data For
8. follow these steps 1 Choose name for your calibration curve standard 2 Choose the wavelength used for measurement or choose from preset wavelengths Bradford 590nm or Lowry 700nm or choose Other and enter your required background normalization wavelength and peak measurement wavelength 3 Place a tip with your blank reagent in to the Picodrop and press the Blank button 4 To begin recording the concentrations of your known standards firstly choose Add Enter the known concentration of your standard in the pop up box and place the pipette tip containing your standard into the Picodrop 5 Press Get this will instruct the Picodrop to measure the absorbance of your standard The absorbance versus concentration will be plotted in a graph in the lower part of the window 6 Upto 5replicates of the same standard can be measured and the average value will be plotted To measure further replicates simply choose the replicate number from the Replicate drop down box and repeat the Get procedure Note it is important that the correct line is highlighted in the Standards Concentration Recorder before changing the replicate number 7 To move on to measuring the second known standard repeat steps 4 to 6 above 8 Aminimum of 2 standards are necessary to draw a linear regression or Polynomial standard line curve Display of calibration standards linear regression In direct measurement detection range depends on th
9. mg ml IgG solution Lysozyme reference Unknown sample protein concentrations are calculated using the mass extinction coefficient of 26 4 at 280 nm for a 1 10 mg ml Lysozyme solution User entered mass extinction coefficient L g cm for a 10 mg ml 196 solution of the respective reference protein a Date amp Time 18 10 2008 17 04 18 10 2008 17 05 ABS 18 10 2008 17 05 ESA 18 10 2008 17 06 Igi 18 10 2008 17 06 LYSOZYME OTHER ear Cyerlays Reset zoom ABS e Pipette 2 5ul of Blank solution into a tip and place the tip and pipette into the plastic pipette holder Click the button marked Blank The software will attempt to blank the spectrophotometer and set the baseline to zero at 280nm A rapid clicking noise will be head for several seconds and the LED on the front of the unit will turn orange e If the unit has not warmed up or if there is residue dust on the lens then the blanking may fail and a message requesting that you re blank will be displayed If this occurs simply try blanking again and if the procure continues to fail try following the lens cleaning protocol e Once the Picodrop has been successfully Blanked then the red Measure button will change to green signifying that you can commence measuring your samples e Insert the pipette with sample and press the Measure button e After a few seconds the concentration in mg ml will appear in the bottom right hand window the spectrum
10. must observe the relevant safety regulations when handling pathogenic material radioactive substances or other substances hazardous to health Operating Environment Please ensure that the ventilation slots of the device remain free to vent at all times A space of at least 10cm should be left around the spectrophotometer The ambient temperature should be between 10 C and 30 C the humidity between 096 and 95 Solvent Compatibility The Picodrop Spectrophotometer unit is compatible with the following solvents Please note that the UVpette Tips should not be used with concentrated solutions of Phenol and Chloroform Acetic Acid dilute Acetone Acetonitrile Benzene Butanol Carbon Tetrachloride Chloroform DMSO Ethanol Ether HCI dilute Hexane HNOS dilute Isopropanol Methanol N Propanol Sodium Hydroxide Sodium Hydroxide Sodium Hypochlorite bleach Toluene INCOMPATIBLE SOLVENT Hydrofluoric Acid and derivatives Instrument Overview The Picodrop is a microlitre spectrophotometer that provides the user with the facility to recover their sample after measurements have been taken It is a full spectrum 230 850nm spectrophotometer which allows for measurements of common laboratory samples such as DNA RNA and protein in small volumes with a high degree of accuracy and precision Samples are contained within patented UVpette pipette tips There is no possibility for cross contamination or ca
11. will be plotted and details displayed in the table below the graph e tis not necessary to save the results as all results are automatically saved to your user project folders Project Selector E Existing Projects sample Mame Date and Time Fiezult BAC O106 Assay 18 10 2008 16 55 0 1 mg mL DNA 50 Measurement 18 10 2008 16 56 0 4 mg mL Picodrop 260 8314 320 11765 Concentration Date amp Time mimi Imm d 0 5 GI Picodrop UL Spectrometer Ready Pipette made Pointer Clear Overlays Reset zoom ABS ma mL Example of BSA measurement Note When using viscous samples it is important to ensure that the sample does not adhere to the outside of the tip Simply wiping the outside of the tip with a piece of clean dry tissue will normally avoid this potential problem Molar Extinction Coefficients vs Absorbance for 196 Solutions Application of a molar extinction coefficient in the calculation yields an expression of concentration in terms of molarity Given the standard equation A ecl where is the extinction coefficient c the concentration and the path length and that Abs is unit less the units defining will determine the units of c If a molar extinction coefficient having units Lmol em is known and used to convert Abs into concentration the obtained molar concentration can be converted using the equation concentration mg mL concentration g L molarity m
12. 7 samples onto the same graph window by simply clicking on the sample displayed in the table under the graph Overlays can be removed by clicking on Clear overlays shown at the bottom of the graph window Absorbance Wavelength YA Podenp i Spexoneter Ready Ppette mode Ponte gt Cen Overlays Bes Zoom gt Pipette or Cuvette Mode It is possible to use Picodrop with either the holder for UVpette tips or the alternative standard 10mm cuvette holder The sample containment mode defaults to Pipette but can be changed to Cuvette by clicking on the button marked Pipette mode shown at the bottom of the screen 55 1 na pL Pipette mode Cuvetke made Pipette mode Pointer Ce Pointer or Cursor option The software defaults to Pointer which allows the user to move the mouse arrow to any point on the graph and view the absorbance and wavelength displayed on the right of the graph indicated as X and Y The Pointer mode also enables the Zoom function To zoom in to an area of interest move the pointer to a position on the graph then click the left mouse button and hold down whilst moving the pointer to another position A rectangle will be drawn Releasing the left mouse button will activate the zoom magnification To reset the zoom to normal click on Reset Zoom shown at the bottom of the graph window Absorbance Wavelength eg me Ss 3450 Ten 3850 mp 3809 m0 Purvai argh nm
13. DNA 33 Measurerr Protein Measureme i dU 12 10 2008 14 34 12 10 2008 14 34 12 10 2008 14 35 12 10 2008 14 35 12 10 2008 14 35 12 10 2008 14 35 12 10 2008 14 35 12 10 2008 14 37 12 10 2008 14 37 12 10 2008 14 37 12 10 2008 14 38 12 10 2008 14 33 60 8 na uL 62 3 nov pl B2 2 ng uL 53 9 na uL E1 7 na uL 5 3 ng uL 1 5 nag uL 3b 2 ng uL 3r nope 3r 5 nope 3r 1 na uL 36 9 na uL Mew Rename View i Print Delete i Export Exporting Data To export detailed test results in an MS Excel format firstly highlight the line or lines to be exported Use shift arrow keys to select multiple lines G i Au e P ETE Eee TO i 2009 E ii dmm WANI YANA WAY i i i WT ik D P o Project Type DNA 50 Measurement User admin Dete time 18 20 2008 17 14 Sample M Sample name X Date Time 1 ocoli w456 erch x552 3 specimen 152 4 specimen 1b3 5 46778 6 research testi research test j 1 3 t 7 5 10 il 12 u Example Excel spreadsheet detailing test results The data export includes the UV Vis absorbance values for each sample at each 5nm wavelength interval Also included are the calculated values for concentration sample name ID and date stamp Nucleic Acid Measurements e Select the relevant nucleic acid mode dsDNA ssDNA RNA Protein UV Vis from within your project folder in the Project Selector window and click the button New under the heading S
14. ICO 1 ul Spectrophotometer USER MANUAL Version 4 0 21 03 1 1 PO Box 188 Saffron Walden CB10 9BA United Kingdom Tel 44 0 131 2080522 info picodrop com www picodrop com All product and brand names used in the document are trademarks or registered trademarks of their respective holders Copyright 2011 Picodrop Ltd Registered in England no 05505772 Table of Contents Safety Warnings and Precautions Potential Safety Hazards Instrumentation Overview On Receipt of Your Instrument Instrument Setup Connection and Loading Software Setting the IP Addresses in a Windows Vista Systems 8 Windows 7 Logging in to software General Use File navigation using the Project Selector page Exporting Data Nucleic Acid Measurements Direct Protein Measurements Indirect Protein Measurements Bradford Lowry Unknown General UV Vis mode Microarray mode General Software Features Printing Maintenance Trouble Shooting Fibre Optic Cable Handing and Limits of Warranty Page Number 10 11 14 15 16 18 19 22 25 27 29 32 35 38 39 40 Safety Warnings and Precautions Please read this section before operating the spectrophotometer Operators of this instrument must be trained in both general laboratory safety practices and the specific safety requirements of the spectrophotometer If the equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impair
15. Instrument List of items included with your Picodrop Spectrophotometer Picodrop Spectrophotometer Unit Pipette Holder with pipette guide collar 1 5mm Allen Key for maintenance Pipette Optional Box of 96 UVpette tips Ethernet Cable 2 Fibre Optic Cables with Connectors 12V Power Cable and Transformer Software Disk User Manual Site The spectrophotometer should be placed on a rigid flat clean surface Make sure that the instrument is completely stable Adequate ventilation is important Make sure there is sufficient space so that the rear and side air slots are not covered and to allow cooling air to circulate freely around the instrument There should be no paper under the device as this may block the ventilation path The unit should always have at least 10cm distance to the next wall or neighbouring instrument Picodrop Ltd spectrophotometer instrumentation is developed for operation in laboratories in which there is normal ambient temperature and no explosive atmosphere The ambient temperature should be between 10 C and 30 C the humidity between 0 and 95 Connect to the electricity and check that the LED light on the front of the unit is illuminated For best results allow unit to warm up for 10 minutes before measuring samples this allows the lamp to stabilise Note the unit can be left connected and switched on all the time This will not damage the unit and only uses power to illuminate the LED The flash lamp is n
16. Printer j Print to file Preferences Find Printer Number of copies 1 E Selection Current Page Pages With the correct printer selected clicking the Print button should start the printing process For details of the other options available and printer troubleshooting refer to the help files on the PC and the specific instructions supplied with the printer selected Example printout Double Stranded DNA d AXI 1 54 2 65 y SUE GU TTA 253 ke 0 11 20 H H SS oa a Wavelength nm QUE TLE TIE Oe O ort UO gor qas gu UO settings Hama Data printed 30 10 2008 22 02 28 Date sample recorded 30 10 2008 21 59732 Maasuramant 52 1 ngu Purity AZGDAZEC 3 333 Absorbance wavelength table 230nm 235nm 240nm 245nm 250nm Z255nm Z260nm 265m ZT nm 275nm 0 256 0 215 0 307 0 464 654 0 628 14042 0 438 0 433 0045 Z lnm zB5nm 290nm 95mm 300mm 305nm Fenm 315nm nmM 325nm 034 0 032 Q048 0 042 0H Aal s3 0 017 DU TaT A nm 335nm 0 050 0 065 Maintenance Cleaning Picodrop spectrophotometers are designed to require a minimum amount of maintenance by the user They can be cleaned using water or a mild laboratory cleaning agent e g ethanol or general lab cleaner The instrument should not come into contact with aggressive solutions Ensure that no liquid enters the spectrophotometers For safety re
17. ample A new window will open which displays a graph and table e Pipette 2 5ul of Blank solution into a tip and place the tip and pipette into the plastic pipette holder e Click the button marked Blank The software will attempt to blank the spectrophotometer and set the baseline to zero at 260nm A rapid clicking noise will be head for several seconds and the LED on the front of the unit will turn orange e lf the unit has not warmed up or if there is residue dust on the lens then the blanking may fail and a message requesting that you re blank will be displayed If this occurs simply try blanking again and if the procure continues to fail try following the lens cleaning protocol Ome veer i Em Propet seo thok Heer fnit une haitis unn gt beck fever unti 0 pount 110 Mack Nove poeu thn lieet peoos un Hack ges Zana Nam Cate edi ree Anna Absorbance Wavelength 260 8805 320912255 Ze 290 0 1 rT tim So 2e 0 200 2020 20 00 239 A40 390 3200 Boo 3800 Wevsengh nn Example of graph window awaiting the blank solution and message indicating that the Blanking was unsuccessful A The Cat is not coreatert Plague ack and ima ne the Geet Lise 306 300 106 26028946 320 12070 1 fm tia fec 390 200 2000 2095 20 MO pn 700 31200 so 300 ra vl enam nn e Once the Picodrop has been successfully Blanked then t
18. asons the device must be switched off and disconnected from the power supply prior to cleaning Quick Clean procedure for sample holder In the event that sample leaks from a pipette tip or dust reduces the light transfer through the pipette holder simply unscrew the silver cable fibres from each side of the silver pipette holder no tools required silver screws should be only hand tight Unscrew the circular base from the tube section Unscrew the single screw on the tube to release the main tube from the bottom tip holder Either soak the holder in hot water with detergent for 30mins and air or drip dry or alternatively simply wash with an ethanol or similar solvent Reassemble and re test instrument If this quick clean procedure does not improve the results please follow the Service Clean procedure described on the following page Important Please note that as the lenses get dirty with use then the stability will decrease and this will accentuate any background noise and small variations between tips To check the light intensity passing through the lenses please login as admin using the password password and then blank and measure the blank In the top left corner of the screen above graph some numbers are quoted for intensity at different wavelengths One of these numbers is described as power band the number displayed should be between 2 and 9 The higher the number the weaker the intensity so cleaning would be rec
19. ch lens with pure water and dry with tissue e Use a cotton tip soaked in acetone to clean and dry the lenses e Remove the round base from the holder by unscrewing e Thoroughly rinse the metal base unit in pure water and then allow to air dry e Re assemble unit and repeat tests as detailed above Connection Lost message This message tells the user that the spectrometer has crashed Disconnecting the power from the spectrometer and reconnecting it should solve the problem There is no need to shut down or restart the software and your data should remain intact unless using the electronic pipette which does introduce a 1mm air gap after the sample Fibre Optic Cable Handing and Limits of Warranty Scope The intent of this note is to define the recommended handling practices for fibre optic cables supplied by Picodrop Ltd and to comment on warranty issues Section 1 Handling Guidelines A B C D E Strength and Proof Testing Do not proof test Strength or proof testing is done by the fibre manufacturers It is a carefully controlled process because it has been found that the more one stresses fibre near its strength limit reduces it overall life time expectation to continue to resist failures Therefore it is specifically recommended that no fibre be proof tested or strength tested except for sample testing in order to gather nominal strength statistics Any overall proof or strength testing is one at t
20. d the absorbance of the dye at its absorbance maximum A max The calculations are based on the Beer Lambert law A E x path length cm x concentration M where E is the extinction coefficient in cm M The absorbance measurements can also be used to determine the concentration of nucleic acid in the sample Optimal labelling for hybridization to microarrays is 1 dye for every 12 20 bases Higher levels of labelling interfere with hybridization Measuring the Base Dye Ratio Measure the absorbance of the nucleic acid dye conjugate at 260 nm A260 and at the A max for the dye Adye Measure the background absorbance at 260 nm and Xmax using buffer alone and subtract these numbers from the raw absorbance values for the sample e To perform these measurements the nucleic acid dye conjugate should be at a concentration of at least 5ug mL Depending on the dye used and the degree of labelling a higher concentration may be required Correct for the contribution of the dye to the A260 reading Most fluorescent dyes absorb light at 260 nm as well as at their max To obtain an accurate absorbance measurement for the nucleic acid it is therefore necessary to account for the dye absorbance using a correction factor provided by the manufacturer CF260 and the following equation Abase A260 Adye x CF260 Calculate the ratio of bases to dye molecules Use the following equation base dye Abase x Edye Adye x Ebase w
21. e and can t be handled as such Any fibre optic cable constructed with quality techniques and materials should survive as long as care is taken when handling it The most precarious moments in a fibre cables life occur during inspection testing and installation It is very easy to exceed the bend radius guideline especially when working with large core cables and performing these tasks
22. e assay here are some examples using the UVpeite tip Note that if you use the 10mm cuvette option then the lower limit will decrease to 5X the detection sensitivity i e 20ug ml from regular Bradford assay Regular Bradford sample range 100 500 ug ml 25 ug ml Mini Bradford sample range 15 50 ug ml 4 ug ml Lowry sample range 0 2 1 0 mg ml 0 01 mg ml General UV Vis Measurements The UV Vis mode displays the entire spectrum split into 2 modes UV 230 360nm and Visible 380 850nm e Select the required UV Vis mode from within your project folder in the Project Selector window and click the button New under the heading Sample A new window will open which displays a graph and table e Pipette 2 5ul of Blank solution into a tip and place the tip and pipette into the plastic pipette holder e Click the button marked Blank The software will attempt to blank the spectrophotometer and set the baseline to zero at normalization wavelength if set A rapid clicking noise will be head for several seconds and the LED on the front of the unit will turn orange If the unit has not warmed up or if there is residue dust on the lens then the blanking may fail and a message requesting that you re blank will be displayed If this occurs simply try blanking again and if the procure continues to fail try following the lens cleaning protocol The UV Vis mode is designed to report the ABSORBANCE at 2 chosen wavelengths There are 2
23. easurements possible for all users before forced to re blank default is 10 maximum recommended is 20 General Use Correct Pipetting Procedure Vortex sample briefly 5 20secs Spin down samples briefly 10 15secs in a microfuge Using a P10 pipette and a UVpette tip pipette your sample A minimum volume of 2 5ul is recommended To minimise solution on the outside of the tip avoid submerging the tip too far below the sample meniscus If necessary Wipe off excess liquid from outside of tip with a dry lint free tissue this is particularly important if using viscous protein solutions Be careful not to touch the bottom of the tip as the sample may be drawn out by the tissue Do not place UVpette tips or your sample too close to heaters or the fan of the PC as heating the tips or sample may result in a rapid contraction in volume once the tip is placed the cooler pipette holder This sample contraction will result in a space or bubble being visible at the bottom of the tip This space may interfere with sample measurement if allowed to rise more than 2mm up the tip It is preferable that the sample tip and pipette holder are allowed to equilibrate to room temperature for 5 minutes before commencing measurement The detection point of the tip is 1 5mm from the end of the tip so it is best procedure to try not to submerge the tip more than 1mm into your solutions otherwise tip wiping may be necessary It is optional whether you decide
24. ed All functions performed within the context of preparing performing and completing a run should be done with caution and care and with general respect both to the instrumentation and to associated chemicals samples and other devices Ensure that anyone involved with the operation of the instrument is instructed in both general practices for laboratories and specific safety practices for this instrument Always place the instrument in a location where if necessary the mains power supply can be disconnected immediately Symbols and Conventions The following chart is an illustrated glossary of the symbols that are used in this manual A CAUTION This symbol indicates a potential risk and alerts you to proceed with caution CAUTION This symbol indicates the presence of high voltage and warns the user to proceed with caution Potential Safety Hazards Electrical Standard electrical safety precautions should be applied Ensure that the proper voltage is supplied before turning the instrument on for the first time The device must be connected to a grounded socket Do not touch any switches or outlets with wet hands Switch the instrument off at the mains supply before disconnecting the AC power cord Unplug the instrument prior to cleaning up any major liquid spills and prior to servicing any of the electrical or internal components Only qualified personnel should perform electrical servicing Hazardous Substances You
25. g on the reference you read the E for single stranded DNA ssDNA is 20 or 30 g cm L while E for RNA is 25 g cm L Extinction coefficients can be used to estimate the concentration of a sample dissolved in a known aqueous volume or to calculate the number of grams or moles of nucleic acid according to the following formulas Determining Concentrations A260 O D 1 Unit for dsDNA 50 ug ml 50 ng ul A260 O D 1 Unit for ssDNA 33ug ml 33 ng ul A260 O D 1 Unit for RNA 40 ug ml 40 ng ul The A320 is used to subtract absorbance due to particles in suspension Direct Protein Measurements e Select the Direct Protein mode from within your project folder in the Project Selector window and click the button New under the heading Sample A new window will open which displays a graph and table e Choose the relevant factor dependent on sample by clicking on the drop down list at the bottom of the graph window The options listed are detailed below Sample Types ABS is a general reference setting based on a 0 196 1 mg ml protein solution producing an Absorbance at 280 nm of 1 0 A where the path length is 10 mm Bovine Serum Albumin reference Unknown sample protein concentrations are calculated using the mass extinction coefficient of 6 7 at 280 nm for a 196 10 mg ml BSA solution IgG reference Unknown sample protein concentrations are calculated using the mass extinction coefficient of 13 7 at 280 nm for a 1 10
26. ge covers the required 600nm wavelength One cursor is normally fixed at the frequently used wavelength for monitoring cell suspensions 600nm while the second cursor can be set to any other wavelength of interest Sample Homogeneity The user must be sure to homogeneously suspend the cells when sampling for absorbance measurements and it is important to read the sample immediately to avoid significant cell settling Vigorous mixing may be required when measuring concentrated samples If the cell suspension is particularly turbid then the Picodrop may not accept the blanking step and as such it may be necessary to reduce the sensitivity of the instrument through the Admin gt Settings see Troubleshooting Section Microarray Dye Measurements e Select the Microarray mode from within your project folder in the Project Selector window and click the button New under the heading Sample A new window will open which displays a graph and table e Pipette 2 5ul of Blank solution into a tip and place the tip and pipette into the plastic pipette holder e Click the button marked Blank The software will attempt to blank the spectrophotometer and set the baseline to zero at normalization wavelength if set A rapid clicking noise will be head for several seconds and the LED on the front of the unit will turn orange If the unit has not warmed up or if there is residue dust on the lens then the blanking may fail and a message
27. he red Measure button will change to green signifying that you can commence measuring your samples e Insert the pipette with sample and press the Measure button e After a few seconds the concentration in ng ul will appear in the bottom right hand window the spectrum will be plotted and details displayed in the table below the graph e tis not necessary to save the results as all results are automatically saved to your user project folders Picodrop Application admin 9g e E J Picodrop Absorbance Wavelength 1 01 0 95 0 55 0 24 AC D I 260 8144 320 11680 0 60 0 54 0 47 D A0 0 33 0 18 0 12 0 06 0 01 230 0 240 0 250 0 27600 2700 280 0 2900 300 0 3140 0 320 0 330 0 340 0 wWwawelength nm Concentration Concentration 4260 4260 AzBD ATHD Date amp Tin 54 0 GI Picodrap pL Spectrometer P Pipette mode Pointer Clear c he Reset Zoom Example results using 260nm wavelength standard Measuring Nucleic Acid Concentration The absorbance of a chemical is a product of its concentration x optical path length x extinction coefficient E Nucleic acids have a peak absorbance in the ultraviolet range at about 260 nm When the spectrophotometer has a path length of 1 cm absorbance optical density O D and O D E x concentration Extinction coefficients vary with the type of nucleic acid Double stranded DNA dsDNA has an E 20 g cm L Dependin
28. he risk of damaging the fibre Specifically proof testing a fibre at or below its bend radius minimum or in any way uncontrolled or unapproved by Picodrop Ltd will void the warranty Bend Radius Do not bend the fibre past the minimum bend radius The rule of thumb regarding bend radius is that the Minimum bending radius for a particular fibre is 300x the cladding diameter assuming the cladding is also glass Twisting Do not coil or twist the cable when spooling un spooling coiling or uncoiling Cables must be handled in a hand over hand fashion at all times Fibre cables should not be handled like rope and coiled or uncoiled by twisting or untwisting one loop at a time Fibre should always be handled in a hand over hand fashion making coils or unrolling coils by moving the fibre in a circle one hand over the other To do otherwise is to induce a twisting stress in the cable and hence in the fibre within the cable Very high stresses can be achieved if a cable is uncoiled incorrectly and damage or weakening can be the result and this type of handling will void the warranty Pulling Do not pull fibre cables by their end fittings And be careful of pulling in general unless your specific cable has been rated to withstand this stress Fibres are usually very strong in direct tension relative to the cross section but when fibres are small it is very easy to break them Other Handling Comments Optical fibre is not wire or rop
29. he tip can occur if the tip is not securely attached to the pipette of if the end of the pipette tip is pushed against the side of the pipette holder when placing the tip in the location hole Re sample and check the liquid column in the tip both before and after sampling If this is satisfactory and the result is still not good increase the sample volume to say 3ul and do the visual checks before and after measurement If results are not satisfactory follow the Quick Clean procedure detailed on the previous page If the results are still not good then the following Service Clean procedure should be adopted as detailed below Service Clean Procedure Detach the Silver fibres by unscrewing from each side of the silver pipette holder Use the 1 5mm Allen key as supplied to loosen the 2 fibres by turning the two sunken screws in the front of the bottom of part of the pipette holder and then unscrew the two fibres away from the holder and attach the LENS EXTRACTION TOOL pictured to right purchased separately to each lens in place of the fibre and pull firmly on the lens extraction tool to pull out lenses Important if the fibres do not come away from the holder easily please seek assistance from Picodrop service team info picodrop com DO NOT PULL ON THE FIBRE OPTIC CABLES Check whether either lens at the end of each cable is wet This happens when excess sample is picked up on the outside of the tips e Wash ea
30. here Edye is the extinction coefficient for the fluorescent dye and Ebase is the average extinction coefficient for a base in double stranded Nucleic acid dsDNA or ssDNA or RNA Note that since the calculation is a ratio the path length has cancelled out of the equation dsDNA Ebase 6600 MWbase 330 ssDNA Ebase 8919 MWbase 330 RNA Ebase 8250 MW base 340 Measuring the Concentration of Nucleic Acid The absorbance values A260 and Adye may also be used to measure the concentration of nucleic acid in the sample N A In order to obtain an accurate measurement for a dye labelled nucleic acid a dye corrected absorbance value Abase must be used In addition for concentration measurements the path length in cm is required The path length of the UVpette tip is 0 9mm but all values are multiplied to display values equivalent to a standard 10mm cuvette N A mg mL Abase x MWbase E base x path length Microarray 13 00 11 69 10 38 8 07 7 76 6 45 V souRqOSgY 5 14 1 21 Wavelength nm Sample Information Name Date printed 05 12 2003 12 34 01 Date sample recorded 05 12 2008 12 28 56 Dye A CY3 cone 714 715 abs 8 541 550nm Dye B CYS conc 548 101 abs 12 218 650nm Submede DNASD result 1214 1 ng uL Cursor A Dnm 0 0004 Cursor B Onm 0 0004 Normalization wavelength nane General Software Features Graph overlays It is possible to superimpose graphs for up to
31. ilar may damage the unit or cause erratic performance Connect the jack plug from the power supply module to the jack socket on the rear of the Picodrop Then switch on the supply The LED on the front of the unit should be illuminated If it does not check all connections are made correctly and that the supply is switched on at the mains switch Connect the instrument to a suitable PC using the short Ethernet cable supplied Powerinput aaV DC E 1 a mg A E sd Note The instrument should be connected directly to the Ethernet port other configurations are not supported To install the Picodrop software insert the CD ROM supplied in the CD Drive of the computer The disc should autorun if it does not please go to Windows Explorer right click on the Picodrop disk and select Autoplay Once the software is installed it is necessary to setup the Ethernet connection following the steps described on the next page Minimum specification for the PC Microsoft Windows XP Service Pack 2 Vista or Windows 7 512Mb RAM 2 8GHz Pentium 4 or 1 6GHz Core Solo or Core Duo A Microsoft amp DirectX amp 9 compatible graphics card with at least 64Mb of on board RAM e g Nvidia GeForce 5900 or better 200Mb of free disc space One Ethernet port If downloading software from the Internet please click on the Setup file The software will install automatically leaving a shortcut on the desktop to the Picodrop interface
32. ol L x molecular weight g mol Examples Proteins and Protein Mixtures with Unknown Extinction Coefficients If no extinction coefficient information exists for a protein or protein mixture of interest and a rough estimate of protein concentration is required for a solution that has no other interfering substances assume epercent 10 Most protein extinction coefficients zpercent range from 4 0 to 24 Therefore although any given protein can vary significantly the average for a mixture of many different proteins likely will be approximately 10 Our software will convert Absorbance at 280nm into mg ml of protein using the formula Indirect Protein Measurements Bradford Lowry Unknown NOTE Typing results into a calibration curve is administrator only function e Select the Indirect Protein mode from within your project folder in the Project Selector window and click the button New under the heading Sample A new window will open displaying the Calibration Explorer table e Choose the relevant calibration curve you want to use from the list or press New to setup a new calibration curve e Once you have chosen your standard from the Calibration Explorer list follow the standard Picodrop procedure Firstly blanking the Picodrop using a tip containing your blanking buffer and then measuring your unknown samples Calibration Explorer To set up a new standard in the Calibration Standards Recorder box
33. ommended for any value above 6 The value displayed as S260 relates to the 260nm energy level this should be between 4 500 and 7 000 counts When the S260 level falls below 4 500 the instrument is still useable but the noise will be higher Please note the latest software is required for this test so please download from website www picodrop com Trouble Shooting Trouble Blanking Blank Inconsistent message If the readings during the blanking stop being linear over the range or there are individual readings outside the 5 limit then the blanking step will be rejected and the user asked to re blank Changing these values is not recommended although for some non homogenous samples e g cell cultures In direct Proteins and some high absorbance saline microarray buffers it may be advantageous to reduce the sensitivity of the unit To reduce the sensitivity log in as Admin and choose the link settings in the top menu Change both the values shown by default as 1 05 to a higher value such as 1 07 The default is 5 so this is why linearity and outlier removal are set to 1 05 If you continue to obtain unexpected or erroneous results you should remove the pipette from the holder and immediately inspect the tip to check that the sufficient sample remains in the tip The liquid column should be at least 2mm in height and should be continuous from the end of the tip i e no air gap at the bottom Leakage from t
34. ot operating unless a sample measurement is made Instrument Set Up Hardware installation If not already pre assembled The pipette holder has been dismantled and disconnected for shipment Fix the pipette holder to its round base by simply screwing it on Assembling the pipette holder Locate the 2 Silver fibre optic cables The fibres have SMA connectors at each end Remove the rubber protectors Connect the cables to the front of the instrument to either one of the SMA connectors at the base of the pipette holder Push the connector into the socket and hand tighten the nut to hold the connector in place Do not use tools to tighten the fixing nut Connecting the cable to the pipette holder Slide the provided PIPETTE GUIDE collar onto the pipette The easiest way to attach the collar is to place the collar in the tall pipette holder and then insert the pipette until the collar has located on the pipette Remove the pipette and the collar will remain attached to the pipette The collar can be easily removed for cleaning if necessary Fully Assembled Picodrop Connection and Loading Software Picodrop uses an external switch mode power supply that will accept any mains voltage from 100 to 240V AC at 47 to 63Hz If the correct mains plug has not been supplied an alternative lead or plug may be fitted If in doubt consult a qualified electrician Use only the power supply module supplied other makes although sim
35. rry over on a sample platform Precious samples can be handled within a sterile environment and are completely recoverable A 2 5ul sample is drawn up directly into the UVpette using a P10 pipette The pipette is placed into the holder which positions the tip through a light beam emitted from a fibre optic cable connected to the tip holder The light source is a pulsed xenon lamp and the light path through the tip is 0 9mm A spectrophotometer analyses the light after it passes through the sample via a 1 dimensional CCD array detector contained within a second fibre optic cable on the opposite side of the holder The instrument is controlled using Picodrop software run from a PC laptop The user can choose to see the data in real time or read and store the data in a tab delimited Microsoft Excel format ONLY USE PIPETTE TIPS MANUFACTURED BY PICODROP LIMITED Consumable re order details Ref PETO1 Interchangeable P10 pipette tip block with lenses fibres Ref CUVO1 Interchangeable 10mm Cuvette block with lenses fibres Ret UVTIPB P10 UVpette pipette 96 boxed tips UV transparent to 230nm Ref UVTIPG P10 UVpeite pipette tips 2 000 loose bagged UV transparent to 230nm Ref PCALO1 GLP PicoCal UV Vis 260nm Calibration Standard fluid 750ul tube Ref P3600L 10 Excel Electronic Pipette 0 5 10ul with adapter for use with Picodrop Ref F144802 Manual Gilson Pipetman Pipette 0 5 10ul with adapter for use with Picodrop On Receipt of Your
36. to use the same tip for both blank and sample or a different tip each time The special ultra low retention tips will ensure zero carryover of blank to your sample and so using the same tip is a safe operation Using different tips for blank and sample will also provide results which meet the published specification but does however introduce an additional variable which may become significant with very low concentration samples i e 10ul ng DNA Recommended pipetting procedure File navigation using the Project Selector page The Picodrop software is structured in a simply project based format To get started follow these steps gt 1 Create a Project folder by clicking on New button shown under the heading project on right side of the Project Selector window Type in a name for your folder 2 Click on your new project folder shown in folder tree on left side of Project Selector window 3 Choose the mode you want to use from the options listed for e g dsDNA ssDNA RNA Direct Protein 4 Start measuring your samples clicking on New shown at the top right side of the Project selector window under the heading Sample Note that if logging in with individual user accounts the user will only be able to view their project folders Thereby providing security against unauthorised access to test results or accidental deletion by other users NOTE All graphs and tables displayed within the software take the actual path length of 0 9mm
37. ways to choose your desired wavelengths NOTE WAVELENGTHS MUST BE CHOSEN BEFORE PRESSING MEASURE otherwise no data will be reported in the table 1 Click on the button Pointer shown at the bottom of the page and choose Cursor then move the crossed lines to the wavelength of interest and right click or press enter Repeat this procedure to choose the second wavelength of interest 2 The 2 wavelengths of interest may also be entered directly by choosing Edit from the options listed when clicking on the pointer button Clicking Edit will display 3 boxes the top 2 boxes allow for the input of 2 wavelengths of interest which be reported on in the report table The lower box allows the user to set a Normalization wavelength This is an optional setting and if set the absorbance value at this wavelength will be deducted from all the absorbance values across the spectrum i e the Normalization wavelength absorbance value is used as the baseline Edit ursors Cursor amp wavelength 00 830 0 S80 0 630 0 680 0 730 0 7800 amp Wavelength nm Cursor B wavelength n Normalization wavelength n Cursors Date amp Time use to cancel values v Pointer Cursor Edit Clear Overlays Reset Zoom Measuring Cell Cultures The UV Vis mode displays the entire spectrum split into 2 modes Extended UV 220 360nm and Standard 380 850nm The standard ran
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