PO Box 188 Saffron Walden CB10 9BA United Kingdom Tel: +44 (0
Contents
1. Figure 1 Assembling the pipette holder Locate the 2 Silver fibre optic cables one is connected to the front of the instrument The fibres have SMA connectors at each end Remove the rubber protectors Connect the cable coming from 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 8 of 8 Figure 2 Connecting the cable to the pipette holder Connect one end of the second fibre to the detection connector at the front of the instrument and the other end to the socket on the pipette holder Figure 3 Fully Assembled Picodrop 9of9 Computer Requirements minimum Microsoft Windows XP Service Pack 2 Operating System 512Mb RAM 2 8GHz Pentium 4 or 1 6GHz Core Solo or Core Duo A DirectX 9 compatible graphics card with at least 64Mb of on board RAM e g Nvidia 5900 or better 200Mb of free disk space One dedicated USB2 port i e not on a hub whether internal or external Software Installation Please install the software before connecting the instrument to the PC Insert the installation disk in the CD drive Please click on the Setup file The software will install automatically leaving the user with a shortcut on the desktop to the Picodrop interface Instrument Start Up Connect the instrument to the PC by inserting the2. dsDNA 50 gt W Auto Range Cursor WaveLength Dye 1 oo BB E BEA EE EE ae 260 280 og u Figure 11 19 of 19 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 reasons 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 black cable fibres from each side of the silver pipette holder no tools required screw 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 20 of 20 Trouble Shooting If you should obtain an unexpected erroneous result you should remove the pipette from the holder and immediately inspect the tip to check that the sufficient samp
3. Picodrop PO Box 188 Saffron Walden CB10 9BA United Kingdom Tel 44 0 131 2080522 L Fax 44 0 207 1173239 info picodrop com www picodrop com Picodrop Microlitre Spectrophotometer Version 1 06 User Manual CE All product and brand names used in the document are trademarks or registered trademarks of their respective holders Copyright 2008 Picodrop Ltd Registered in England no 05505772 VAT GB 881 3758 91 Table of Contents Page Number Safety Warnings and Precautions 3 Potential Safety Hazards 4 Instrumentation Overview 6 On Receipt of Your Instrument 7 Instrument Setup 8 Software Installation 10 General Use 12 Blanking 13 Nucleic Acid Measurements 14 Protein Measurements 16 UV Vis Measurements 17 Microarray Dye Measurement 18 Maintenance 19 Trouble Shooting 20 2of2 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 impaired 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 anyon
4. USB2 cable into the socket at the rear of the Picodrop Power up the spectrophotometer by connecting the 12V power supply to the input jack The orange light at the front of the Picodrop will indicate that the instrument is on Start the program by double clicking on the Picodrop icon on the Desktop 10 of 10 Picodrop Software Application Tabs Nucleic acids wecro array Uv Visible Proteins Help Sample ID Input Reading button Real time reading button Store Save Sample Blanking button Sample Type pees ess E E E Result Display Reference wavelength in nm Figure 5 Picodrop software Main Screen Function Buttons amp Text Boxes Application Tabs allows the user to switch applications Sample ID Input allows automatic sample numbering or custom input Read button pressed each time a measurement is required Real time reading button On Off displays the measurements taken in real time Store result saves the current read results Results are stored to a temporary file Save stored results saves data in the temporary file permanently The data is stored in MS EXCEL format compatible with spreadsheets Blanking button allows the user to take a blank reading of buffer or water prior to sample reading Sample Type selector User selects the specific pre set nucleic acid or protein setting Result Display Sample concentration determine
5. 0 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 1mm 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 only supplied as replacement spare part Ref UVTIPB P10 UVpette pipette 96 boxed tips UV transparent to 230nm Ref UVTIPG P10 UVpette pipette tips 2 000 loose bagged UV transparent to 230nm Ref PCALO1 PicoCal UV Vis 260nm Calibration Standard fluid 75ul tube Ref PCALO1 GLP PicoCal UV Vis 260nm Calibration Standard fluid 750ul tube GLP NIST Traceable certified 6 of 6 On Receipt Of Your Instrument List of items included with your Picodrop Spectrophotometer Picodrop Spectrophotometer Unit Pipette Holder 1 5mm Allen Key for maintenance P10 Pipette Optional Box of 96 UV Irradiated UVpette tips USB Cable 2 Fibre Optic Cables with Connectors 12V Power Cable and Tra
6. 5 0f 15 Protein Measurements e Select the Proteins application tab e Select the sample type e g BSA e Tick the Auto Range box if you want the axis of the graph to set limits automatically e Blank the Picodrop e Insert the pipette with sample and press the Start Reading button e After a few seconds the concentration in mg ml will appear in the bottom right hand window and the spectrum will be plotted e Click on the Store Sample button and either continue with measurements or save the stored results by clicking on the Save Stored Samples button n aloja 10 mm Absorbance hond sUMSRSHSESRIAERERS Z mo ao zwo oO moO a MOO oO mo wo w o Raference Wavelength f280 abs 2610 auto A 280 10 mm Pathf26 18 260 280 0 62 mg m SSS Figure 9 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 16 of 16 Sample Types A general reference setting based on a 0 1 1 mg ml protein solution producing an Absorbance at 280 nm of 1 0 A where the pathlength 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 1 10 mg ml BSA solution IgG reference U
7. Thoroughly rinse the metal base unit in pure water and then allow to air dry Re assemble unit and repeat tests as detailed above 21 of 21
8. bance to zero The blank solution is usually the reagent which you have used with your samples Select a new tip and pipette 2 5ul of blank solution Carefully insert the whole pipette and tip into the holder as illustrated below Figure 7a c Correct insertion of the pipette tip assembly into the holder Click on the Blanking button on the software main screen The indicator light on the front of the instrument will flash whilst the measurement is being taken Blanking is complete when the sample button changes colour from grey to green To check that the blanking is acceptable i e with an Absorbance at 260nm lt 0 05 at the wavelength of interest click on Start Reading A line at the bottom of the screen represents the blanking over the entire spectrum Note It is important to repeat the blanking at regular intervals e g after 5 minutes or 10 20 measurements 13 of 13 Nucleic Acid Measurements e Select the Nucleic Acids application tab e Select the sample type dsDNA ssDNA RNA e Tick the Auto Range box if you want the axis of the graph to set limits automatically e Blank the Picodrop e Insert the pipette with sample and press the Start Reading button e After a few seconds the concentration in ng ul will appear in the bottom right hand window and the spectrum will be plotted e Click on the Store Sample button and either continue with measurements or save the stored results by clicking on
9. ce 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 0 and 95 4 of 4 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 HNO3 dilute Isopropanol Methanol N Propanol Sodium Hydroxide Sodium Hydroxide Sodium Hypochlorite bleach Toluene INCOMPATIBLE SOLVENT Hydrofluoric Acid and derivatives 5 of 5 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 carry 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 P1
10. cent 10 concentration in mg ml The relationship between molar extinction coefficient emolar and percent extinction coefficient percent is as follows emolar 10 epercent x molecular weight of protein Still other sources provide protein absorbance values for 0 1 mg ml solutions as this unit of measure is more convenient and common for protein work than percent solution This variation in reporting style underscores the importance of carefully reading stated values to be sure that the unit of measure is understood and applied correctly Examples A 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 percent range from 4 0 to 24 0 5 Therefore although any given protein can vary significantly from epercent 10 the average for a mixture of many different proteins likely will be approximately 10 Note the Picodrop software assumes the user enters the epercent value cited above Our software will convert Absorbance at 280nm into mg ml of protein using the formula Protein Concentration in mg ml Abs at 280nm corrected to 10mm pathlength epercent x 10 17 of 17 UV Vis Operation Select the UV Visible application tab Select the ref
11. d from the absorbance reading AutoRange Automatically rescales the y axis limit to suit the maximum absorbance AutoSave Automatically SAVES read results to the MS EXCEL spreadsheet after each measurement thereby removing need to press Store sample after each measuresment 11 of 11 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 Wipe off excess liquid from outside of tip with a dry tissue this is particularly important if using viscous protein solutions 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 will interfere with sample measurement It is preferable that the sample tip and pipette holder are allowed to equilibrate to room temperature for 5 minutes before commencing measurement Figure 6 Recommended pipetting procedure 12 of 12 Blanking the Picodrop Before sample measurements are made it is necessary to measure a blank solution to set the background absor
12. e 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 3 of 3 Potential Safety Hazards Electrical Standard electrical safety precautions should be applied e Ensure that the proper voltage is supplied before turning the instrument on for the first time e The device must be connected to a grounded socket e Do not touch any switches or outlets with wet hands e Switch the instrument off at the mains supply before disconnecting the AC power cord e 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 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 devi
13. erence wavelengths Blank the Picodrop Insert the pipette with sample and press the Start Reading button e After a few seconds the result will appear in the bottom right hand window and the spectrum will be plotted e Click on the Store Sample button and either continue with measurements or save the stored results by clicking on the Save Stored Samples button Picodrop Ruckekc Acid Spectroscope Pre Release VLO eic Ac ll x Nucleic Acids Micro Array UV Visible Figure 10 18 of 18 Microarray Dye Measurements e Select the Microarray application tab e Select the dyes using the scroll arrows beside the Dye 1 and Dye 2 text boxes e Blank the Picodrop e Insert the pipette with sample and press the Start Reading button e After a few seconds the result will appear in the bottom right hand window and the spectrum will be plotted e Click on the Store Sample button and either continue with measurements or save the stored results by clicking on the Save Stored Samples button j nucleic Acids Micro Array UV Visible Proteins Help 1 j i i i Micro Array Spectra gt E 3 i i y j i f 4 E tt ki 400 420 440 460 480 S00 520 540 S60 S80 600 620 640 660 680 700 720 740 Wavelength nm Cursor MicroArray Sample Dy i Real Time Off Store Sample Save Stored Samples Blank
14. le 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 the 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 cleaning procedure should be adopted as detailed below 1 Detach the Silver fibres by unscrewing from each side of the silver pipette holder 2 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 pull gently the two lens holder screws from each side of the holder 3 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 4 Wash each lens with pure water and dry with tissue 5 Use a cotton tip soaked in acetone to clean and dry the lenses 6 Remove the round base from the holder by unscrewing 7
15. nknown sample protein concentrations are calculated using the mass extinction coefficient of 13 7 at 280 nm for a 1 10 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 gm 1cm 1 for a 10 mg ml 1 solution of the respective reference protein Molar Extinction Coefficients vs Absorbances for 1 Solutions Application of a molar extinction coefficient in the calculation yields an expression of concentration in terms of molarity A emolar molar concentration However many sources including the reference cited above do not provide molar extinction coefficients Instead they provide absorbance A280nm values for 1 1 g 100 ml solutions measured in a 1 cm cuvette These values can be understood as percent solution extinction coefficients percent having units of g 100 ml 1 cm 1 instead of M 1cm 1 Consequently when these values are applied as extinction coefficients in the general formula the units for concentration c are percent solution i e 1 1 g 100 ml 10 mg ml A percent percent concentration If one wishes to report concentration in terms of mg ml then an adjustment factor of 10 must be made when using these percent solution extinction coefficients i e one must convert from 10 mg ml units to 1 mg ml concentration units A per
16. nsformer Software Disk User Manual ae Oe ee 2 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 not operating unless a sample measurement is made 7 of 7 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
17. the Save Stored Samples button Picodrop ul Spectrophotometer Pre Release VEIO a lox Nucleic Acids proteins Help Reference Wavelength gt 260 260 320 10 mm Path 18 29 260 280 1 80 fies 280 320 10 mm Path 10 35 foo no u B Abs 260 230 5 18 869 FF Auto Range 320 10 mm Patho os pess Figure 8 Example of dsDNA measurement Measuring Nucleid 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 Depending on the reference you read the E for single stranded DNA 14 of 14 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 33yg ml 33 ng ul A260 O D 1 Unit for RNA 40 ug ml 40 ng ul The A329 is used to subtract absorbance due to particles in suspension 1
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