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User Manual - White Bear Photonics

1. U3 Cruzer Micro USB 2 0 FD USB 2 0 Mass Storage USB Device USB Device USB Device USB Device USB Device K 2 Ocean Optics USB4000 USB DISK USB DISK 2 0 gt USB Flash Disk USB Flash Drive USB Mass Storage Device USB Tool USB Tool USB2 0 HandyDrive USB20 Camera v1i65w 51 item s 4 Selected Only click on an entry that includes the name Ocean Optics before you choose Description SanDisk U3 Cruzer Micro USB De PNY USB 2 0 FD USB Device SAMSUNG HD154UI USB Device USB Device USB Device Hamamatsu TM CCD Series Hamamatsu TM CCD Series Hamamatsu TM CCD Series Ocean Optics USB4 000 Easy Disk USB Device USB DISK 2 0 USB Device General USB Flash Disk USB Device Philips USB Flash Drive USB Device USB2 0 USB Flash Disk USB Device AT SYSTEM USB Tools USB Tool FUJITSU HandyDrive300 USB De USB20 Camera hp 165w USB Device Device Type Mass Storage Mass Storage Mass Storage vendor Specific Vendor Specific Unknown Unknown Unknown vendor Specific Mass Storage Mass Storage Mass Storage Mass Storage Mass Storage vendor Specific vendor Specific Mass Storage vendor Specific Mass Storage Connected No No No No No No No HirSoft Freeware http www nirsoft net DER usb idsisno Uninstall selected devices from the File menu of the USBDeview application If you are not one hundred percent certain whether to
2. According to Beer s Law an extinction coefficient is a constant for a given substance dissolved in a given solute and measured at a given wavelength It is an intrinsic property of a substance that will be the same for differing samples of the same substance Conventional spectrophotometers usually have a minimum pathlength of 10 mm 1cm but the pathlength of the TLDAocean is much less than this To comply with Beer s Law and convention the TLDA ore calculates a result for a sample based on a 1 cm pathlength TLDA soe does this using a mathematical model that is based on the actual pathlength of the light through the sample within the TLDAgcean instrument This small pathlength feature of the TLDAocean instruments design gives the TLDAocean the ability to measure samples that are 1 Many times more concentrated than a conventional spectrophotometers upper detection limit 2 Have avery high absorbance value 3 Much lower in volume than conventional spectrophotometers Note The actual pathlength on the TLDAocean instrument is dependant on the volume of sample placed on the plinth It is therefore very important to input the correct volume loaded on the plinth in to the software interface and pipette accurately 1 5 5 Nucleic Acid Quantitation Purines and pyrmidines in nucleic acid show maximum absorbance of UV light around 260 nm eg dATP 259nm dCTP 272nm dTTP 247nm Nucleic acids are known to have a maximum absorption at 2
3. Fig 2 5 STEP 7 The TLDAgcean IS NOW connected and ready for use Fig 2 5 15 Drop Technology Version 1 3 2 0 Installation 2 2 Software Installation WARNING The spectrometer must be connected via a USB cable to the computer before the TLDAsop software can be loaded onto the PC The requirements to install TLDAsoft are as shown below Requirements PC with 32 bit or 64 bit Windows 8 7 Vista or XP Spectrometer from Ocean Optics USB4000 USB2000 or HR4000 Free USB port 2 0 TLDAsopt File tlda_setup exe 8 8MB Approximately 35MB available space on a hard disk drive C Administrator rights on your PC NOTE To enable all functions of TLDAsop the user must register with Drop Technology www droptechnology com They will then receive by email a software registration key based on the serial number of their spectrometer supplied 2 2 1 Software Installation To properly install the TLDA software l Close all programs and make sure that the Spectrometer is connected via the USB cable into the computer Make sure any previous versions of TLDAsot have been uninstalled from your computer see section 2 2 2 to uninstall previous versions of TLDAsoft Insert the operating software CD or USB key in the CD drive or USB slot of the PC The software installation menu should appear automatically Click on tlda_setup exe TLDA ot application software If this menu does not appear choose My Computer
4. You must then press the accept button to store the dark spectrum data in memory as dk file The measure button is now enabled Fig 4 12 STEP 9 Once the blank is completed open the sample chamber If desired the sample can be retrieved using the Drop Technology Pipette Alternatively the sample area can be cleaned using lint free absorbent wipes Fig 4 12 These or similar wipes are recommended by Drop Technology for their highly absorbent and their lint free properties This simple wiping with a lint free tissue should be all that is required to sufficiently minimise sample carryover between successive measurements Version 1 3 Drop Technology 52 4 0 General Operation Fig 4 13 STEP 10 Pipette the drop of sample solution onto the raised centre of the quartz plinth Fig 4 13 Refer to Fig 4 8a and Fig 4 8b to ensure that the drop pipetted is suitable Fig 4 14 STEP 11 Press Measure on the TLDAsote to measure the absorbance of the sample drop An absorbance spectrum is displayed covering the relevant wavelength range 27 2128 24 4915 21 7702 2 19049 4 o i 2 16 2277 13 6064 oO daf a 10 8851 If you are happy with the absorbance spectrum press Accept If not click Measure again to record a new E E Dama absorbance spectra Alternatively you may click on Decline which will bring you back to the intensity spectrum and all aspects of the software
5. 0 92628 PASS 1 x Baseline Correction at 700nm Lambda3 0 45337 0 46165 0 43568 0 50410 0 47210 0 88832 0 90240 0 90331 0 91091 0 91371 Sample Name Version 1 3 PASS 2 x Baseline Correction at 700nm 3 0 Software The previous steps are repeated for 5 individual drop measurements of RM1 The five spectra are shown colour coded and overlayed on the screen with the absorbance data After 5 readings have been accepted TLDAsofte will automatically calculate the mean and standard deviation for each wavelength which is displayed at the bottom of the table of data Once you double click in the table of data the data table will fill the screen TLDAsote will state whether the TLDAocean Instrument has passed or failed The test can be repeated with the second reference fluid if desired If the second reference standard RM2 is run you must blank the system again and pipette five drops of the RM2 standard The data will be populated on the screen and a pass or fail status displayed In the event of failure see Section 6 3 Troubleshooting Drop Technology 34 3 0 Software 3 2 2 10 Point Validation Check A 10 point validation check is an all inclusive check that ensures that the whole system is performing optimally It takes longer to execute than a 5 point check as it requires 10 repeatable points for full validation This thorough check may be carried out by a service engineer
6. 6 0 Appendix e Inspect pipettes daily for damage to the nose of the barrel where the tip is fitted or any other obvious damage If there is an issue have it serviced because it is unlikely to be fit for purpose e Store pipettes vertically using a pipette holder This prevents any liquids that have sneaked into the barrel of the pipettes from getting any further inside and corroding them Use the correct technique for the sample type being pipetted See forward and reverse techniques outlined below Forward Pipetting Press the operating button to the first stop Dip the tip into the solution just under the liquid surface to a depth of 2 3mm and slowly release the operating button Wait 1 2 seconds and withdraw the tip from the liquid touching against the edge of the reservoir to remove excess liquid Dispense the liquid onto the plinth by gently pressing the operating button to the first stop and then after a short delay press the operating button to the second stop This action blow out will empty the tip Release the operating button to the ready position Ready Position l 2 Suitable for Standard liquids aqueous and nucleotide solutions Genomic DNA amp PCR products First Stop Second Stop Reverse Pipetting Press the operating button to the Second stop Dip the tip into the solution just under the liquid surface 2 3mm and slowly release the operating button Wait 1 2 seconds and withdraw the tip fr
7. The spectrum covers the 200nm 750nm range 0 578786 0 482322 O 3g55a7 n A9798 10mm Absorbance AU 0 192329 Dgs 200 300 400 S00 600 700 B00 Wavelength nnr Version 1 3 Drop Technology 36 3 0 Software Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS Information 5 Pt Performance Check 10 Pt Validation Replicate Lambda 1 0 69875 0 69119 ABSORBANCE E Lambda 2 Lambda 3 E Reference RMI z 0 52688 0 55224 fa pa Bd 0 56242 0 54799 Sample Name RMi x Baseline Correction at 700nm 1 01109 0 909985 0 808875 5 0 707766 w 0 606656 S 5 0 505547 g 0 404438 0 303328 0 202219 0 101109 ye Gy Transmitted Light Drop Analyser Weacune 500 Wavelength nm Settings Calibration DNA RNA Proteins UV VIS Information 5 Pt Performance Check 10 Pt Validation Lambda 1 0 69875 0 66015 0 69167 0 74199 0 66508 0 66138 0 71579 0 64920 0 69757 0 66288 ABSORBANCE Lambda 2 Lambda 3 j Reference RM1 0 56242 0 54799 0 53321 0 52406 0 53849 0 51688 0 56486 0 52081 0 51949 0 51465 0 51048 0 50949 0 53063 0 50706 0 50170 0 51530 0 52589 0 52216 0 49990 0 49026 Sample Name RM1 2237 lt FAIL x Baseline Correction at 700nm Drop Technology The previous steps are repeated for 10 individual drop measurements of the reference material The absorbance spectra are sho
8. to view the contents of the CD or USB key Double click on the file named tlda_setup exe On the Welcome window click Next Ed Installing TLDAsoft epa Welcome to the TLDAsoft Installation This setup program will install TLDAsoft on your computer Click Cancel if you do not want to install this application Click Next to continue the installation WARNING This program is protected by international copyright law and treaties Unauthorized reproduction or distribution of this program or any portion of it may result in severe civil and criminal penalties and will be prosecuted to the maximum extent of the law Drop Technology Cancel Version 1 3 Drop Technology 16 2 0 Installation 3 Please read the License Agreement carefully Before you can proceed to the next window by pressing Next you have to agree to the terms and conditions J Installing TLDAsoft co mE License Agreement To proceed with the installation you must accept this License O Agreement Please read it carefully TLDAsoft Beta Version 4 2 1 This software is only compatible with 32 bit or 64 bit Windows 7 Vista and XP DROP TECHNOLOGY LTD SOFTWARE LICENSE AGREEMENT This Software License Agreement Agreement is made as of Januarv 2012 I agree with the above terms and conditions I do not accept the agreement Drop Technology lt Back Cancel 4 The Choose Destination Lo
9. wavelength needs to be known it can be selected here The wavelength can be selected by using the up down arrows to the right of the wavelength box or clicking in the wavelength box and manually entering in the value The absorbance value displayed will correspond to the wavelength selected The user selected wavelength and absorbance value are not used in any calculations The nucleic acid purity factors A260 230 ratio and the A260 A2g0 ratio are calculated by the TLDAsopepackage and displayed The nucleic acid concentration in ng ul is displayed based on its absorbance at 260nm and the selected constant An absorbance spectrum is displayed covering the wavelength range 200 400nm If a reading outside this wavelength range is required the UV Vis tab should be used Version 1 3 3 0 Software 3 4 Protein Tab 3 4 1 Absorbance Assay 280nm The measurement of proteins is carried out using this tab It is based on the absorbance of proteins at a wavelength of 280nm This absorbance assay is fast and convenient since no additional reagents or incubations are required No protein standard need be prepared and it does not require the generation of a standard curve This App displays the UV spectrum 200 400nm the absorbance at 280nm and calculates the concentration mg ml The absorbance value at 280nm is approximated to a 1 cm pathlength within the software Transmitted Light Drop Analyser Setti
10. 0x1022 MI hex format Manufacturer Name Insert manufacturer name Device Name Ocean Optics USB4000 mS Cere Version 1 3 Drop Technology 18 2 0 Installation 9 The information about the USB device driver has now been collected and must be stored somewhere on the PC You can freely choose a location but the file name extension inf must not be changed Save in L My Documents E amd 4 jia64 My Recent license Documents My Albums IE My videos x86 My Documents 8 My Computer File name Save as type inf files inf My Network 10 Before you click Install Now as shown on the screen shot below make sure that you have chosen the correct device for which the USB device driver will be installed The most obvious indicators are the Vendor ID Ox2457 and the Device description Ocean Optics USB4000 as in the example below Please click Install Now E libusb win32 Inf Wizard o e Information Vendor ID 0x2457 Product ID 0x 1022 Device description Ocean Optics USB4000 Manufacturer Insert manufacturer name Install Now 11 The files for the USB device driver are now been stored in the system folders of the Windows operating system Please wait this might take some time Installing Driver Installing Driver 19 Drop Technology Version 1 3 2 0 Installation 12 When the installation process is finished successfully the window below pops up on the sc
11. Check 10 Pt Validation ABSORBANCE Replicate Lambda 1 Lambda 2 Lambda 3 Sample Name x Baseline Correction at 700nm 65 000 98 500 52 000 45 500 an 39 000 Fa 32 500 2 26 000 19 500 Measure 13 000 6 500 0 500 Wavelength nm Settings Calibration DNA RNA Proteins UV This tab contains two sub tabs 5 Pt Performance Check 10 Pt Validation e 5 pt Performance check e 10 pt Validation The specific protocols for both procedures are contained within the Help tab These protocols should be read thoroughly prior to calibration and can be referred to at any stage during the process by clicking the Help button NOTE Make sure the plinth has been cleaned before beginning calibration 31 Drop Technology Version 1 3 3 0 Software 3 2 1 5 Point Performance Check A 5 point performance check requires a minimum of 5 repeatability tests that can be regarded as a quick pre measurement approval This check will establish that the TLDAoccean instrument is performing to specifications and will give confidence in subsequent measurement results Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS Information q TLDA so z oe 5 Pt Performance Check 10 Pt Validation ABSORBANCE a Replicate Lambda 1 Lambda 2 Lambda 3 Ez Sample Name IRM x Baseline Correction at 700nm 65 000 58 500 52 000 45 500 F 39 000 A 32 500 26 000 J 19 500 i
12. Measure 13 000 6 500 0 Eveatuayt 3 tbe tS che de ag oh nat wnt of tent ky to oF by tuo gh Syeda d 200 300 400 500 600 700 800 Wavelength nm 2 TENTION NET To carry out a 5 point performance test Settings Calibration DNA RNA Proteins UV VIS Information IN D Ason 5 Pt Performance Check 10 Pt Validation z yy you must first enter a Sample Name for tt ets tantes the reference tests going forward fa i Sample Name x Baseline Correction at 700nm Sample Mame RM Blank Measte You must then blank the instrument To do so pipette a drop of the blank solution onto the plinth and press Blank BLANK is now displayed on the screen in between the blank and decline buttons Blank Blank You must then press the now enabled accept button to store the blank spectrum data in memory as bkc file Version 1 3 Drop Technology 32 3 0 Software Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS Information TLD As ft DC 5 Pt Performance Check 10 Pt Validation ABSORBANCE 2 9 Replicate Lambda 1 Lambda 2 Lambda 3 z g x Baseline Correction at 700nm Blank DARK Discard Measure j AA nan 280 300 320 340 Accept Wavelength nm Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS Information TLDAson 5 Pt Performance Check 10 Pt Validation A
13. The TLDAocean is ideally suited to applications where sample is limited samples are highly concentrated or have a high absorbance and speed and analysis of the sample is important e The principal of operation of the Drop Technology TLDAocean is patent protected Fig 1 1 TLDAccean Accessory Part Number DTO600 100209 TecRRSPocY Tra T L DA ocean r NSmi e Mitteq Light Drop Analy Fig 1 2 Fibre Optic Cable to carry light from the light source to the drop via the Fibre Optic Connector on the top of the TLDA Part Number DT0800 300101 Fig 1 3 Drop Technology 10uL Pipette O 5uL 10uL range Part Number DT0820 300107 Version 1 3 Drop Technology 2 1 0 Introduction Fig 1 4 Fig 1 5 a Fig 1 5 b Fig 1 5 c o 3 Drop Technology Fig 1 4 Box of 96 Pipette Tips Part Number DT0820 300108 Fig 1 5 Drop Technology Accessory Kit Part number DTO600 100204 Cleaning and Reference Standard kit combined shipped initially with the instrument Fig 1 5 a Drop Technology Plinth Cleaning Solution Part Number DT0810 300112 Fig 1 5 b Drop Technology Reference standard kit including the standards Drop Technology RM1 Drop Technology RM2 Drop Technology Blank Solution Part Number DT0810 300105 can also be shipped individually See Section 5 4 Fig 1 5 c Cleaning Tool Part Number DTO900 200120 Version 1 3 1 0 Introduction Fig 1 5 d Fig 1 5 d 8mm Spanne
14. e Negative values these values could result if an incorrect solution was used as a blank Alternatively these values could arise due to fluorescence of a dye in the solution 1 5 8 Protein Quantitation Protein quantitation is performed at 280nm on the TLDAocean system Absorption of radiation in the near UV 280 nm by proteins depends on the concentration of the amino acids with aromatic side chains present in the sample such as Tyrosine and Tryptophan but also to a lesser extent Phenylalanine and disulfide bonds Readings do not require the generation of a standard curve as is the case with colorimetric methods of quantifying proteins such as the Bradford Lowry BCA and Biuret assays NOTE Extreme care should be taken to eliminate nucleic acids from the sample before reading protein samples as the extinction coefficients of nucleic acids in the 280nm region may be as much as 10 times that of protein at their same wavelength and hence a few percent of nucleic acid can greatly influence the absorbance and concentration result In the implementation of Beer s law for protein quantitation the extinction coefficient is generally but not always referred to as a molar extinction coefficient The molar extinction coefficient of a peptide or protein is related to and calculated by its amino acid composition The molar extinction coefficients of the amino acids Tryptophan Tyrosine and Cysteine at 280 nm are generally accepted to be 5540
15. nm Baseline Correction The TLDAoean system automatically incorporates a Baseline Correction this normalises the data at 320nm i e the X Baseline Correction at 220nm absorbance value at 320nm is deducted from all absorbance values at all wavelengths This is the data plotted to the screen and displayed in the necessary information boxes This can be disabled so that the raw absorbance data is recorded Sample Name An appropriate sample name ought to be Sample Name DNA200_1 inputted here Sample Type The user must select the type of nucleic acid being measured The options that appear in the drop down list are sample Type Extinction Coefficient i e dsDNA double stranded DNA Nucleic Acid 7 e ssDNA single stranded DNA e RNA e Nucleic Acid for other nucleic acids If this is selected the user has to enter the constant value between 15 and 150 The constant value is the inverse of the molar extinction coefficient and this will be set by the type of sample being tested Wavelength If the absorbance at a particular Version 1 3 Drop Technology 38 3 0 Software psorbance Au Wavelength nrm 39 Ratios 260 230 Ratios Concentration Concentration nol Graph 7 37192 6 63473 5 89754 2 5 16034 w 2 4 42315 Ge p 5 3 68596 H 4 T 2 94877 amp 2 21158 no 1 47438 0 737192 39 260 280 300 320 Wavelength nm 340 260 280 Drop Technology
16. on top of the body Fig 5 14 Drop Technology 58 5 0 Maintenance Fig 5 15 Fig 5 16 N gt E p ad pr XK TecRRSPocY HNO Tran T L D A ocean r SMitted Li ght Drop Analys 59 STEP 9 Screw the centre screw back fully Fig 5 15 Screw the fibre back into the SMA connector Fig 5 16 and tighten another 30 with the spanner provided STEP 10 The TLDAccean is now ready for use Drop Technology Version 1 3 6 0 Appendix The TLDAocean is Compatible with most solvents used in a laboratory Solvent Material Quartz Acetal Plinth Plinth Housing Acetic Acid dilute R NR Acetone R R Benzene R R Butanol R R R Resistant Carbon Tetrachloride R R LR Limited Resistant Chloroform R R NR Non Resistant DMF Dimethyl Formamide R NR Ethanol R R Ether R R HCI dilute R LR Hexane R R lsopropanol R R Methanol R R Sodium Hydroxide dilute R R THF Tetrahydrofuran R R Toluene R LR Version 1 3 Drop Technology 60 INSTRUMENT TYPE SAMPLE SIZE PATHLENGTH LIGHT SOURCE DETECTOR TYPE WAVELENGTH RANGE SPECTRAL RESOLUTION ABSORBANCE ACCURACY DETECTION LIMITS MAXIMUM CONCENTRATION TYPICAL REPRODUCIBILITY 96 REPLICATES TLDAocean 1ul to 4ul Dependent on sample size Pathlength determined from mathematical physical model and verified experimentally Pulsed Xenon CCD array 200 750 nm 1 5 nm lt 2 2 0 ng ul dsDNA 0 05
17. replace the plinth as required Version 1 3 Drop Technology 56 5 0 Maintenance Fig 5 5 NOTE Laboratory gloves must be worn at this point AP T lif lA pent a Unscrew the SMA connector that connects via fibre optic cable from the light source ae to the lid of the instrument Fig 5 5 STEP 2 Unscrew the centre screw fully and remove Fig 5 6 STEP 3 Lift off the lid of the TLDAocean to reveal the plinth underneath Fig 5 7 Fig 5 8 STEP 4 Using the tool provided Fig 5 8 Fig 5 9 Place the tip of the tool under the lip of the plinth and gently lift out the old plinth Fig 5 9 D x D TECRRSPocY 57 Drop Technology Version 1 3 Fig 5 10 Fig 5 11 TecRRRPosY Th T LDA Ocean anami lye ittad Light Drop A Fig 5 12 Fig 5 13 JY TECRRSLOGY Fig 5 14 Version 1 3 5 0 Maintenance STEP 5 Open the box that contains the new plinth Fig 5 10 Try to avoid touching the quartz plinth directly Invert the new plinth and place over circular indentation on the top of the TLDAocean body Fig 5 11 STEP 6 Press the new plinth firmly into place and ensure that it is fitted securely Fig 5 12 STEP 7 Lift off the attachment that came with the new plinth If the plinth is fitted correctly this attachment should lift off easily revealing the new plinth underneath Fig 5 13 STEP 8 Place the lid of the TLDAocean back
18. same drop or repeat measurements on another sample of the same solution are required For Example DNA200 will default to DNA200_1 once the Accept button has been pressed Blank A blank must be carried out before measurements on any sample are taken When taking the blank place a drop of deionised water or the relevant buffer depending on the application on the plinth close the lid and press Blank See section 1 5 Blank The blank file is a measurement of the intensity of light versus wavelength It may be useful to refer to the blank file at a later point during sample measurement The blank file gets automatically saved to the file directory it is assigned the sample name but with the file extension bk i e okd for dna work and bkp for protein work To locate the blank or any other file open the File Manager Decline The decline button declines either the intensity or Decline absorbance spectrum displayed on the screen 25 Drop Technology Version 1 3 Measure Measure Accept Reset Help Shutdown Version 1 3 3 0 Software The measure button sends the command to the system to take a spectral reading Once the button has been pressed an absorbance spectrum will appear on the graph Measure can be pressed more than once until the user is happy with the spectrum This button accepts the generated absorbance spectrum and saves it to
19. test because of the dominance of this surface tension force over the gravitational force The gravitational force is symmetric and therefore so is the micro drop about its vertical axis e Upon closing the lid of the TLDAocean the light source and spectrometer are aligned so that the light path between them is vertical The light is focussed as it passes through the sample drop on the plinth The pathlength of the drop is directly related to the volume of the drop selected e The design of the TLDAoccean eliminates the need for cuvettes both standard size and low volume cuvettes as in conventional spectrophotometers The cleaning of cuvettes is time consuming and can introduce errors The TLDAocean allows for rapid clean up with no measurable sample carryover e Due to the small sample size it has the capability to measure highly concentrated or highly absorbing samples without dilution 1 Drop Technology Version 1 3 1 0 Introduction e The TLDAoccean system is designed for a wide range of applications from nucleic acids and protein quantification to any general UV Visible measurements e The Drop Technology TLDAocean unit is an accessory instrument designed to be used in conjunction with an Ocean Optics Spectrophotometer and an Ocean Optics light source both with a fibre SMA905 connector e The Drop Technology accessory enables the user to measure microlitre drop samples lt 4ul with a high degree of accuracy and precision e
20. the system memory with the file extension dna or prt i e dna for dna work and prt for protein work Note This action also saves the samples intensity data in the form sg i e dsg for dna work and psg for protein work This icon rests all samples to factory default settings should the user wish to return the instrument to its default configuration This button resets the graphical display i e a new blank must be saved for absorbance spectrums to be acquired Help buttons are present in each tab to provide supplementary assistance to the user depending on the application This shuts down the TLDAgofte program This must be done before the TLDAocean is switched off Drop Technology 26 3 0 Software 3 2 Settings Tab This software tab contains the basic user defined settings for the TLDA ofte Users should take care to check the Settings tab prior to taking any measurements to ensure that all fields represent the desired setting values gt Transmitted Light Drop Analyser Settings Calibration DNA ZRNA Proteins UV VIS Information q TLDA son Set Mon 07 January 2013 11 15 01 Save Folder Sample Volume ul 3 0 Yv Integration Time ms 5 Average 10 Boxcar Width 10 a Drop Technology www droptechnology com Time and Date The Time and Date on the TLDAocean is set by the manufacturer prior to shipment of the unit but it can Mon 07 January 201
21. was used in the DNA RNA module in the previous session Version 1 3 Drop Technology 8 1 0 Introduction 1 5 6 Purity and Ratios Nucleic acids have a broad peak at 260nm and so they can be measured accurately and easily provided that there is no source of foreign contamination affecting the peak 7 37192 6 63473 _ 5 89754 Z 5 16034 Fig 1 Screenshot of pure nucleic acid read with no visible source of protein contamination at 280 nm g E 4 42315 o 5 3 68596 9 fa T 2 94877 amp 2 21158 no 1 47438 0 737192 pee a sl Lem el eae al cS es Wma aa cn E a fee Decl Waal ey med eo 200 220 240 260 280 300 320 340 360 380 400 Wavelength nm It is common for nucleic acids to be contaminated with other molecules hence we can assess the degree of purity of the nucleic acids by examining the absorption at other wavelengths rather than 260nm Due to the fact that these contaminating molecules such as protein and polysaccharides have their own known absorption maxima the absorbance at other wavelengths is often compared to the absorbance at 260nm Due to the fact that proteins have an absorbance maximum at 280nm and many polysaccharides absorb at 230nm the A260 A2g0 and A260 A230 ratios may be used for their detection This is the standard method to determine if the nucleic acid sample is contaminated The equations for calculating these ratios take into account the effect of turbidity at 320 nm Neither nucle
22. when calibrating the machine 2352 Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS Information TLDA Soft ot 5 Pt Performance Check 10 Pt Validation ABSORBANCE 9 Lambda 1 Lambda 2 Lambda 3 ae Reference E Sample Name RM1 x Baseline Correction at 7F00nm 65 000 58 500 52 000 45 500 B 39 000 i 26 000 19 500 oe pease 6 500 0 500 Wavelength nm Firstly the user must select from the Reference drop down menu either RM1 or RM2 The specifications for both reference KMI 7 saa standards are stored within the software memory and thus the software itself can determine whether the TLDAocean system passes the validation or not You must then blank the Instrument To do so pipette a drop of the blank Blank Bei solution onto the plinth and press Blank BLANK is now displayed on the screen in between the blank and decline buttons i L ACCADE You must then press the now enabled accept button to store the blank spectrum data in memory as bkv file Drop Technology Version 1 3 3 0 Software Transmitted Light Drop Analyser Seinge Calbraton DNA RNA Rote WIS Inman lh ESY O DARK is now displayed on the screen in 5 Pt Performance Check 10 Pt Validation aoe between the blank and decline buttons Lambda 1 Lambda 2 Lambda 3 Reference RM1 You must then press the accept button Sample N
23. 1480 and 134 respectively These values are then weighted with their amount within the protein and summed to give the molar extinction Emolar Of that Protein 11 Drop Technology Version 1 3 1 0 Introduction Emolar NAxX5540 nBx1480 nCx134 where nA the ratio of Tyrtophan residue relative to the other two nB the ratio of Tyrosine residue relative to the other two nC the ratio of Cysteine residue relative to the other two 5540 1480 and 134 the amino acid molar extinction coefficients of Tryptophan Tyrosine and Cysteine at 280 nm Applying this molar extinction coefficient and the value for the measured absorbance of a solution yields an expression for the molar concentration A Molar Concentration E molar when L 1cm In practice many suppliers of standard proteins do not provide molar extinction coefficients Instead they provide absorbance Azgonm values for 1 1 g 100 ml solutions measured in a 1 cm cuvette These values can be described as mass extinction coefficients Emass or percent solution extinction coefficients having units of g 100 ml cm instead of M cm or Lmol cm or ml ug cm as referred to earlier Consequently when these values are applied as extinction coefficients in the general formula the units for concentration C are in terms of percent solution i e 1 1 g 100 ml 10 mg ml Percent Concentration The relationship between molar extinction coeffici
24. 3 11 15 01 E be adjusted by the user at any point To set the Date and Time press the Set button on the left hand side of the Time and Date field this stops the clock Press OK to accept the prompt window that appears Place the Cursor in the Time and Date field and highlight the increment to be changed Use the up arrow and the down arrow to increase or decrease the increments of time and date Press the asterisk again to accept the time and date and restart the clock from this point onwards Ensuring that the Time and Date is set correctly is important because by default data files are saved in chronological order This will lead to confusion identifying data if the time and date is incorrect NOTE The time and date does not reset itself to take account of daylight saving time 27 Drop Technology Version 1 3 Save Folder Save Folder Help Reset a Sample Volume Sample Volume ul 3 0 Integration Time Integration Time ms 5 Version 1 3 r 3 0 Software This Field details the location for which the data is to be saved If the user wants to organise sets of measurements into a folder for ease of identification afterwards they can input a folder name here Once a folder name is created all sample data will be saved into this folder until the folder is changed or returns to default The default for this is an empty field By default files automatically get saved to the ro
25. 60nm which is an average of the absorption of the individual nucleotides 7 Drop Technology Version 1 3 1 0 Introduction Beer s law is applied to the absorbance value recorded in conjunction with the selected inputted extinction coefficient and the pathlength of light through the sample to determine the concentration in ng uL Therefore the amount of light absorbed is directly proportional to the concentration of nucleic acid in the sample The manipulation of Beer s Law gives the concentration in the form A C EL Therefore C concentration measured in mg mL A Absorbance AU mass extinction coefficient uL ng cm L pathlength in cm The generally accepted mass extinction coefficients for the most abundant nucleic acids are Double stranded DNA 0 02 uL ng cm Single stranded DNA 0 03 uL ng cm RNA 0 025 uL ng cm Hence for a 1cm pathlength the optical density at 260nm equals 1 0 for the following solutions A 50ng ul solution of dsDNA A 33ng ul solution of ssDNA and A 40 ng ul solution of RNA TLDAson calculates the concentration ng uL from the equation zZ Kk Where A260 absorbance value at 260nm for a 1cm path length 1 Factor EL Therefore the Factor for the common nucleic acids based on a 1cm pathlength will be dsDNA 50 ng uL ssDNA 33 ng uL RNA 40ng uL The TLDA ofte package automatically defaults to the last mass extinction coefficient that
26. BSORBANCE H L Replicate Lambda 1 Lambda 2 Lambda 3 az 0 69242 0 55161 0 57136 ja z Sample Name RM x Baseline Correction at 700nm 1 06392 5 0 957529 4 0 851137 4 0 744745 4 2 0 638353 47 S 4 5 0 53196 G lt 0 425568 amp 0 319176 H f 0 212784 roor ro ooo A 400 500 600 Wavelength nm 1 06392 0 957529 0 851137 0 744745 0638353 0 53196 0 425568 E 0 319176 j 0 212784 0 106392 0 m Absorbance AL 33 Drop Technology DARK is now displayed on the screen in between the blank and decline buttons You must then press the accept button to store the dark spectrum data in memory as dkc file The measure button is now enabled Wipe off the blank drop using a lint free wipe and pipette the first drop of the Reference Solution RM1 on the sample plinth and press Measure If you are happy with the absorbance spectrum press Accept If not click Measure again to record a new absorbance spectra Alternatively you may click on Decline which will bring you back to the intensity spectrum and all aspects of the software will be enabled This will store the data for the absorbance spectrum in memory as a cal file The corresponding intensity data will then also be stored as a csg file The table of data will populate as the reference measurements are taken The Absorbance of the sample drop is retrieved at three different pre co
27. Drop Technology It s the light inside Transmitted Light Drop Analyser TLDAocean User Manual Version 1 3 Drop Science a technology The Decisive Step Towards the Future tiny drops yield oceans of data www droptechnology com Drop Technology It s the light inside www droptechnology com Thank you for purchasing the Drop Technology TLDAocean Your instrument has been manufactured with the utmost care and has been tested prior to dispatch For technical support please contact your local representative or go to www droptechnology com Please ensure that you know your instrument model and individual serial number before you get in contact NOTE It is strongly recommended that you read this manual fully prior to using your instrument REGISTRATION Please register your product Drop Technology periodically publishes information relating to this product We can alert you of these updates if you are on our user list All information supplied to Drop Technology is completely confidential You can register at www droptechnology com Drop Technology Ltd Tallaght Business Park Whitestown Dublin 24 Ireland Tel 353 1 4523293 Fax 353 1 4523967 Email info droptechnology com www droptechnology com CONTENTS AL SOS IAG FO CHUNG CIO tel scons aatetcctedeitaitanc uaa EE 1 OD POOL sty SIN ON eae cta i a set ascetics rsetealect ornare alent al ol aaiecac seine ataciessasted ar atsinte teadea
28. E Basic gt Ulead Systems Ulead Instant Viewe SRD APIO m Avizdvd r oil Adobe Reader 8 i Outlook Express a AviSynth 2 5 T AcaFilter ESS windows Media Player Haali Media Splitter A z aS Windows Messenger m xvid c Windows Movie Maker A Imoburn p Bink and Smacker A Prism Video File Converter mag T MATLAB 7 1 I Microsoft Office Tools E Microsoft Project a RCG E e Potytec E T NCH Software Suite 14 The TLDAsot icon on the Windows start menu starts the TLDAsof application Please choose the Information tab of this application and check whether the spectrometer is linked to the application software or not In the example below it states Yes for a USB4000 device This indicates that the spectrometer is properly communicating with the software In the case of a No in the Linked column the device is either not connected to a USB port of the PC or the communication between the software and this particular device is corrupted Please contact the customer service if you require any help i E Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS Information Device Linked Identification Refresh USB4000 Yes USB4C03194 Now USB Key Yes MassStorage Administrator Name Password TLDAsoft Copyright Drop Technology Beta Version 4 2 1 Drop Technology www droptechnology com Version 1 3 Drop Technology 20 2 0 Installation 15 If the connecti
29. TLDAocean system The Drop Technology 10 uL pipette is recommended for use with all TLDA instruments NOTE Evaporation of the sample may become an issue with the use of certain samples If the samples have a high vapour pressure or a long timeframe is used measures must be taken to ensure that they do not evaporate during the reading as this would have a detrimental effect on the result The TLDAocean design incorporates a circular channel highlighted in red below in the plinth enclosure that can be filled with liquid to create a saturated atmosphere within the closed testing chamber This evaporation well can be filled with the sample liquid or deionised water depending on the requirements Fig 4 1 Saturation channel highlighted on plinth The desired liquid once pipetted into the well and left to equilibrate will act to slow down the evaporation rate within the chamber and lead to better results Version 1 3 Drop Technology 48 4 0 General Operation 4 3 Taking a measurement ready NOTE Before taking any measurements make sure that you have your blanking solution sample solution pipette pipette tips and lint free tissues Delays during the measurement process could affect results if samples have a high vapour pressure Set Mon 07 January 2013 11 15 01 E N 1 Sample Volume ul 3 0 Integration Time ms 5 s Average 10 Boxcar Width Ya 5 Drop Technology www droptechno
30. ame RM1 Baseline Correction at 700nm ee to store the dark spectrum data in memory as dkv file 52 000 4 45 500 4 DARK p 39 000 4 s2 500 asm The measure button is now enabled 13 000 4 easune 6 500 4 0 ee 200 220 240 260 280 300 320 340 360 380 400 Accept Wavelength nm Wipe off the blank drop using a lint free wipe and pipette the first drop of the reference solution onto the sample plinth and press Measure If you are happy with the absorbance spectrum press Accept If not click es O Measure again to record a new Transmitted Light Drop Analyser ABSORBANCE i E See absorbance spectra Alternatively you ej a may click on Decline which will bring Sample name RN NC centre carton at 700mm you back to the intensity spectrum and 0 964643 ol all aspects of the software will be 0 771715 enabled Sm J bog Pa By pressing Accept the data for the a absorbance spectrum will be stored in memory as a val_ file The corresponding intensity data will then also be stored as a vsg file 0 69875 0 56242 0 54799 as A The table of data will populate as the reference measurements are taken The absorbance of the drop is displayed at three different pre configured wavelengths 0 064543 5 0 863179 D71713 0 57525 An absorbance spectrum for each drop sample is displayed on the screen
31. ar to the drop shown in Fig 4 8 a If the drop is similar to the drop shown in Fig 4 8 b It must be removed and a new sample must be pipetted NOTE If the drops are consistently like those shown in Fig 4 8 b it may be due to contamination of the plinth In this case cleaning or changing of the plinth may be necessary See section 5 0 Maintenance STEP 6 Rotate the lid back so that it clicks into place with the light source directly over the plinth Fig 4 9 NOTE Do not rotate the lid more that 120 at one time Always allow for the lid to click into place before moving it again Drop Technology Version 1 3 4 0 General Operation Fig 4 10 STEP 7 Press Blank on the TLDAsogeinterface The blank spectrum will appear on the screen BLANK is now displayed on the screen in between the blank and decline buttons 5 077 empmemem ger meer orem pe er ee me 58 500 4 Blank 52 000 45 500 3 39 000 F 32 500 Deene You must then press the now enabled accept button to store the blank spectrum data in memory 26 000 19 500 13 000 ease 6 500 TTT 200 220 240 260 280 300 320 340 360 380 400 Wavelength nm This will save the blank measurement to memory with the Sample name given with the extension bk i e bkd for dna work and bkp for protein work STEP 8 DARK is now displayed on the screen in between the blank and decline buttons
32. at these two wavelengths calculated by TLDAsofte and displayed The protein concentration in mg ml is calculated based on its absorbance value at 280nm and the relevant selected extinction coefficient An absorbance spectrum is displayed covering the wavelength range 200 400nm If a reading outside this wavelength range is required the UV Vis tab should be used Drop Technology 42 3 0 Software 3 5 UV Vis tab This tab enables the TLDAocean to Operate as a conventional spectrophotometer Any measurements in the ultraviolet visible range of light 200 800nm can be measured Absorbance can be found at up to three different wavelengths The tab also displays the maximum and minimum absorbance at their respective wavelengths across the 200 800nm spectrum V Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS Information TLDAson Wavelength1 nm Wavelength2 nm Wavelength3 nm 230 420 S 650 2 Absorbancel AU Absorbance2 AU Absorbance3 AU Y Y MIN nm E Sample Name a Baseline Correction nm at 630 gt vj 65 000 58 500 52 000 45 500 3 39 000 32 500 Decline 2 26 000 19 500 13 000 peanae 6 500 0 200 220 240 260 280 300 320 340 360 380 Wavelength nm Baseline Correction Baseline correction can be enabled at any wavelength within the range for UV Vis measurements This normalises the data at the wavelength selected i e the abso
33. ator Name eeeeeeee Registration Code TLDAsoft Copyright Drop Technology Beta Version 4 0 0 Drop Technology www droptechnology com 19 Enter in the 30 digit registration key and then click the activate button 20 Your TLDAsop package should now be ready for operation with your TLDA 21 Drop Technology Version 1 3 2 0 Installation 2 2 2 Software Uninstallation Uninstall can be chosen from the Windows start menu and the process starts with the message as shown below eee Welcome to TLDAsoft Uninstallation 8 Uninstalling TLDAsoft Are you sure you want to uninstall TLDAsoft This uninstall process will remove TLDAsoft from your computer Click Cancel if you do not want to remove this application Click Next to continue the uninstall process Drop Technoloay i j Cancel Please be really sure that you want to uninstall TLDAsom from your PC before you click Next When you have clicked the Next button the process cannot be stopped again Only a re installation of TLDAsom Can return it to a working application To uninstall the TLDA software 1 By pressing Next in the Welcome window shown above starts the application USBDeview This tool allows you to uninstall any USB driver So be careful to choose the correct one from the list Currently connected and activated USB devices show a green dot on the left hand side USBDeview File Edit view Options Help xg eooo PRacan Device Name
34. aveverencenradaceseaceweditesignwwaseeycareveiaancdactwedens 57 OO APDE 9 0 camera ey tecntsertne ner ie eee agent he ee ee er Reno Eee er aera s 60 6 SOWENT COMID Atl DIN LY scxteiietatetin tend eaes ensaauinaesiahbelsnceiasnaiad A EN 60 6 2 TELDA o PECCI ON S ae E O 61 MOOS DOONUN aeae NE AE E O TTN 62 64 Parte that Reduire RE Order seenen E ERE 63 625 Pipettine TCI CQUC nenna a a a E E a NA 64 COAN SU rss ete TEE A EAS 67 1 0 Introduction 1 0 Introduction The TLDAcceane is a simple to use UV Visible instrument based on the physics of drops which leads naturally to more reliable instruments e The abbreviation TLDA stands for Transmitted Light Drop Analyser The TLDA instrument can be purchased as either an accessory as is the case with the TLDAcsoe and the TLDAcceane Or as a complete integral laboratory instrument which is the case with the TLDA ve e In traditional UV Visible spectrophotometers a sample is placed within a cuvette whereas within the TLDA instrument the cuvette is replaced with a microvolume drop sample 1 4ul e t relies upon the TLDA patented technology This is based onthe physics of surface tension which holds the microvolume drop sample in place The sample is in the form of a sessile drop that in the TLDAoceane adheres very strongly to the edge of the hydrophilic plinth Importantly the drop shape is virtually constant regardless of the liquid under
35. cation screen appears If there is a folder C droptech on your machine already you should move its content to a different location The TLDA installer will overwrite any content in an existing folder C droptech or creates a new one in order to save the program and data files in it Click the Browse button to customize your installation location or click the Next button to accept the default location and continue J Installing TLDAsoft o ma Installation folder The folder C droptech will be created automatically A Please press Next to continue the installation process Destination folder C droptech Browse Space required 21 07 MB Space available 170 37 GB Drop Technology 5 Please wait until the TLDAsof installer has copied all the files to your hard disk Installing TLDAsoft co es Installing Files Copying TLDAsoft files to your computer O To stop or pause the installation process click Cancel Directory C droptech images File banner800x148 jpq Drop Technology Cancel 17 Drop Technology Version 1 3 2 0 Installation 6 The window above disappears automatically and the libusb win32 Inf Wizard starts with the preparations of the USB device driver You should connect a spectrometer to a USB port of your PC if you have not done so already Please click Next FS libusb win32 Inf Wizard Lo eta Information This program will create an inf file for you
36. cquire the relevant Administrator Name and Password For access to these please contact your local distributor or Drop Technology Drop Technology 46 3 0 Software Drop Technology makes periodic upgrade of its software TLDAsofte These upgrades are available for download at www droptechnology com The software upgrade can be downloaded onto your PC or saved onto a USB key which is then connected to the PC The updated software must be downloaded directly onto the PC or saved onto a USB key which is then connected to the PC You must firstly uninstall the previous software version see section 2 2 2 and then install the updated software as per instructions in the manual NOTE It is important to follow the manual instructions or else driver files etc could be deleted mistakenly 47 iDropTechnology Version13 4 0 General Operation The top of the TLDAgcean body can be rotated by 120 to reveal the sample chamber Clockwise or counter clockwise rotation will open left handed or right handed sample chambers to aid both left and right handed users The size of the drop is defined by the user The Drop Technology Pipette must be set to the required sample volume 1 4 uL Drop Technology recommends an optimum drop size of between 2uL and 3uL Pathlength is dependant upon drop size The Pathlength volume relationship is determined by a mathematical physical model of the
37. created before or after the installation You or your other applications may need them C droptech data test bku C droptech data test dku C droptech data test1 usg C droptech data testl uvs C droptech data test2 usg C droptech data test2 uvs C droptech init info C droptech init log C droptech usbdeview USBDeview cfg Delete Delete All Drop Technology 23 Drop Technology Version 1 3 2 0 Installation 5 Click the Finish button when the uninstallation is complete 39 Uninstalling TLDAsoft b la x TLDAsoft has been successfully uninstalled Click Finish to complete the uninstallation Drop Technoloay If the USB device driver was uninstalled you can now disconnect the spectrometer and use the vacant USB port for different USB devices or you can connect the spectrometer to a different USB port and use it with another software application Version 1 3 Drop Technology 24 3 0 Software Sample Name This field is present in all of the measurement tabs DNA RNA Proteins and UV Vis It allows for specific sample identification By double clicking in this field a virtual keyboard will appear to assist the inputting of the desired name sample Name Samplet_1 If the sample name is not changed for new tests it will automatically add an underscore and number on to the end This is convenient for time saving in instances when repeat measurements on the
38. d 49 iDropTechnology Version13 4 0 General Operation x Settings Calibration DNA RNA Proteins UV VIS Information TLDA Son f e api nm Wovelgar rm Wavelger rm i U V VI S a e 420 S 650 a Absorbance1 AU Absorbance AU Absorbance3 AU Tm a g e Baseline Correction Sample Name No 1 Baseline Correction nm at lz E Sa m p e N a m e 65 000 sa m e Wavelengths 1 2 and 3 52 000 4 45 500 4 3 39 000 4 3 F 32 500 5 i gt 2 26 000 4 19 500 4 Measure 13 000 aaa ELLA 400 500 Wavelength nm STEP 3 On the TLDAoccean reveal the preferred sample chamber according to your preference for left or right handed TechRLocy operation Tra TL DA ocean ef Smitted Light Drop AnaS F 7 4 6 gt P I g e e i Fig 4 7 STEP 4 Pipette the drop of the relevant blanking solution onto the raised centre of the quartz plinth Fig 4 7 NOTE Ensure that there are no bubbles in the sample NOTE The Drop Technology pipette should be used in accordance with good pipetting technique see section 6 5 Version 1 3 Drop Technology 50 4 0 General Operation Fig 4 8 a Fig 4 8 b Fig 4 9 ee STEP 5 The drop must be a stable sessile drop for accurate results to be obtained The drop Should stand upright on the plinth be proud of the quartz surface and be of uniform shape The drop must be simil
39. do delete the Ocean Optics device driver please contact customer service Version 1 3 Drop Technology aD a 2 0 Installation 2 If you decided to uninstall the device driver as shown in the previous step another window pops up This gives you the opportunity to cancel the uninstallation process or proceed with that choice USBDeview Da s BE wr A Do you want to uninstall the selected devices Please exit the USBDeview application by choosing Exit on the File menu of the window shown in step 1 whether you have uninstalled the device driver or not 3 At this point in time you have pressed Exit on the File menu of the USBDeview application and the uninstallation process continues with the deletion of the TLDAsot files Please wait this can take a little while 29 Uninstalling TLDAsoft co tse Uninstalling P Removing TLDAsoft application from your computer p To stop or pause the uninstalling process click Cancel Deleting files B Drop Technology Cancel 4 The next window allows you decide whether you want to delete some or all of your data log and configuration files which have accumulated during the time when TLDAsoftt was installed on your PC If you delete all of these files the folder droptech is removed from the PC otherwise it will remain 239 Uninstalling TLDAsoft gt fecal Files not deleted Files not deleted by the uninstallation process O The following files were
40. e TLDA on package automatically measures calculates and records the A260 A280 ratio for each sample so the purity can be determined via the ratio or by sight in some cases by viewing the graph A user can interpret a graph such as Fig 2 as showing an overshadowing of the 260nm peak with a higher absorbance peak at 280nm This implies that there is a higher concentration of protein than nucleic acid in the sample which in extreme cases could lead to the 260 measurement being affected as well as a poor A260 A280 ratio Version 1 3 Drop Technology 10 1 0 Introduction 1 5 7 Contaminants affecting results e As well as protein and peptide contamination at 280nm results can be affected by the presence of other impurities e Absorption at 230 nm can be caused by contamination by phenolate ion thiocyanates and other organic compounds e Phenol contamination Phenol is commonly used in nucleic acid purification but it can significantly throw off quantification estimates Phenol absorbs with a peak at 270 nm and an A260 Azgo ratio of 1 2 Nucleic acid preparations uncontaminated by phenol should have an Agz o 280 ratio of around 2 Contamination by phenol can significantly contribute to overestimation of DNA concentration e Particulate contamination Absorption at 330 nm and higher indicates particulates contaminating the solution causing scattering of light in the visible range The value in a pure nucleic acid sample should be zero
41. e tis good practice to periodically clean the sample plinth to prevent the possibility of contaminant build up The cleaning interval will depend again upon the nature of the samples being measured and the amount of use the instrument receives Fig 5 1 STEP 1 NOTE Laboratory gloves should be worn at this point Place one piece of cleaning cloth over the head of the tool Fig 5 1 STEP 2 Fix the circular ring securely over the cleaning cloth and the tool head Fig 5 2 Fig 5 3 STEP 3 Place one drop of cleaning fluid onto the plinth Fig 5 3 55 Drop Technology Version 1 3 5 0 Maintenance STEP 4 Rub the surface gently in alternating circular movements Stop when the surface appears dry and all the fluid has been absorbed Fig 5 4 STEP 5 If a small number of samples are tested or the samples are of a low concentration deionised water provides sufficient cleaning However after cleaning with the cleaning fluid deionised water must then be used to rinse the surface When using deionised water repeat STEPS 1 4 but replace the cleaning fluid with deionised water e If the recommended cleaning procedures are followed the plinth should only need to be changed once a year e t may be the case that there is an evident scratch on the surface of the plinth or that an extremely corrosive sample has damaged the quartz surface finish In this instance it is up to the user s discretion to
42. echnology TLDAocean accessory 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 use This will prevent the possibility of failure as a result of internal condensation Contact your supplier immediately if you experience any unexpected difficulties with the Drop Technology TLDAocean NEVER LOOK DIRECTLY INTO THE BEAM OF UV VIS LIGHT FROM THE LIGHT SOURCE It could cause permanent or temporary blindness ENSURE THAT THE LAMP IS SWITCHED OFF PRIOR TO CONNECTING AND DISCONNECTING THE FIBRE OPTIC CABLE This will avoid eye damage caused from exposure to the light source NOTE BEFORE MEASURING SAMPLES ALLOW THE LAMP IN THE LIGHT SOURCE TO WARM UP Recommended time varies according to the lamp NOTE If the solutions to be tested with the TLDAocean instrument are flammable corrosive toxic or otherwise harmful all due care and attention must be practiced while working with such substances to minimise risk to the operator and the instrument i e good laboratory practice and consultation with the Solvent Compatibility Table in Section 5 1 If the MSDS for dangerous samples can be attained it is recommended that it is read and fully understood prior to using the sample Drop Technology Version 1 3 1 0 Introduction e If the accessory is used in a manner not specified or in environmental conditions not appropriate for its safe ope
43. ed and No means that the device has not yet been detected Identification lists the serial number of individual components parts This is particularly important in the case of the spectrometer and light source as the manufacturers may need to refer to these at a later date NOTE The spectrometer serial number is needed to produce your software registration key which is required for the software to be operational Version 1 3 Refresh Refresh Mow Help Reset a Administrator Name and Password Administrator Name Password Version 1 3 3 0 Software When now is clicked within the Refresh section the software is prompted to scan the system for devices and updates the status of all devices found It may be used to detect the presence of a new device such as e A USB storage key If the device details are not present on the list after clicking refresh try clicking refresh again After retrying if it still does not pick up the USB details you should try an alternative USB key e f the software does not pick up the identification number of the spectrometer click refresh again or reboot the system If it is still not present you should contact the distributor This provides information for the user in regards to all functions in the information tab This button resets the TLDAocean system to its default settings For certain functions to be carried out the user must a
44. el at 45 degrees Pipette against the sidewall or into the liquid that s already there e End users should check the accuracy of pipetting by dispensing 100uls onto a fine balance The mass of the droplet you make should be around 0 1 g Now do the same thing 10 times and record the masses obtained If the variation is more than 0 5 the pipette may need to be looked at or more practice is required e Pre wet the tip When dispensing liquid from the pipette a coating of the sample is left on the tip making the expelled volume slightly less than it should be Pre wetting the tip before pipetting will help this Just draw up the liquid into the pipette then dispense it back into the original vessel The coating is now on the tip so when liquid is drawn up again and dispensed into the receiving vessel none of it will be lost to wetting This is only recommended for volumes greater than 10uls Maintain inspect and store pipettes properly e Have pipettes serviced every 6 12 months or more frequently depending on the accuracy required The service should include re calibration greasing of the moving parts and replacement of any worn out seals or other parts It s best to have this done by an experienced pipette engineer Pipettes should be wiped down with 70 ethanol before they are used every day Pipettes should be disassembled and cleaned internally and externally with 70 ethanol on a weekly basis 65 Drop Technology Version 1 3
45. ent Emolar and mass extinction coefficient Emass is as follows E mass MW oa a AMEE molar ji 0 Where MW the molecular weight of Protein KDa 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 e g one must convert from 10 mg ml units to 1 mg ml concentration units 10 Concentration in mg ml NOTE As there is such variation in reporting styles of proteins it is very important to carefully read stated vales to be sure that the unit of measure is understood and applied correctly Version 1 3 Drop Technology 12 1 0 Introduction Although the same revision of the equation is used for Beer s law as was employed for nucleic acids Convention dictates slightly different unitary nomenclature within the law be utilised for protein concentration so proteins are measured in the units of mg ml Therefore C concentration measured in mg ml or g 100ml A Absorbance AU molar extinction coefficient M cm or mass extinction coefficient Lg cm L pathlength in cm The TLDA on package calculates protein concentration for the end users in mg ml It has 3 predefined user selectable coefficients e BSA Unknown Bovine Serum Albumin protein concentrations are calculated using the mass extinction coefficient of 6 67 at 280nm for a 1 10mg ml BSA solution e IgG Unknown Immunoglobulin G concen
46. epeatedly pipetting cold samples the first dispensed volume is always larger than expected but subsequent pipetting with the same tip gave the correct volume The reverse was true for hot samples the first disoensed volume was smaller than expected Their solution was simple dispense the first volume back into the original vessel then start pipetting e Use a sensible pipette for the volume to be dispensed The accuracy of a pipette decreases as the dispensed volume approaches the minimum volume of the pipette So for dispensing 15uls for example a 1mL pipette would be terrible a 200ul pipette not so good and 20ul pipette ideal We recommend the Drop Technology P10 pipette for use with TLDA instruments as the instrument has been optimised for use with this pipette e Use well fitting tips Poorly fitting tips allow air to escape when drawing up and dispensing leading to inaccurate results 6 6 Warranty All TLDAgcean Spectrophotometers and accessories manufactured by Drop Technology are warranted against manufacturing defects in parts and labour for a period of one year This is a return to base Warranty and a proof of date of purchase should be provided with returned goods Please contact Drop Technology or the distributor before you return an item 67 Drop Technology Version 1 3 Drop Technology Its the light inside Drop Technology Ltd Tallaght Business Park Whitestown Dublin 24 Ireland Ph 353 0 1 452 3297 Fa
47. ese fields will populate Minimum Absorbance As with the maximum absorbance the TLDAsofte interface automatically displays the wavelength and absorbance values at which MIW AU ri the sample had the lowest absorbance After measurement these fields will populate Graph 6 99634 6 2967 The graphical representation of results from the sample in this tab is displayed from 200nm 800nm 5 59707 Z 4 89744 o 4 1978 is 3 49817 o Oo 2 79853 amp 2 0989 no 1 39927 0 699634 o Ll 200 300 400 500 600 700 800 Wavelength nm Version1 3 DropTechnology Me 3 0 Software 3 6 Information The top half of this tab contains the details and status of connected devices The bottom half of this tab contains tools that facilitate the upkeep of the TLDA instrument Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins Device Linked Identification LISB4000 Yes LISBSHO5663 USB Key No NA Administrator Name UV VIS Information iL L D A Soft Refresh Now Password TLDAsoft Copyright Drop Technology Beta ersion 4 0 0 Drop Technology Connected Devices Device Linked Identification USB4000 Yes USB4H05663 USB Key No N A 45 Drop Technology www droptechnology com The name of the Device is listed under Device section The Linked section shows whether or not the device is connected and responsive Yes means it is connect
48. ic acids nor proteins absorb light at 320nm so this wavelength in used to correct the readings and remove the turbidity effect Turbidity causes light to be scattered and affects quantification of DNA and RNA The Turbidity absorbance may also be measured at 340 nm for Protein measurements Ay o _ A 60 7 A3997340 A560 _ A460 a A207340 Ago Aso E A320340 A230 A439 7 A339 340 Where A230 Absorbance of a sample at 230nm A260 Absorbance of a sample at 260nm A2g0 Absorbance of a sample at 280 nm A320 340 Absorbance due to turbidity A320 for nucleic acids or A340 for protein e An Az60 A280 ratio of 1 8 is generally accepted as pure for DNA e An Az60 A280 ratio of 2 0 is generally accepted as pure for RNA e The Axgo A230 ratio should be greater than the Argo Azg0 ratio The A260o A230 ratio should not be lower than 1 8 in any case The A260 A230 ratio usually falls in the range of 1 8 2 2 If the ratio is much lower than expected it may indicate the presence of contaminants which absorb at 230nm e A gt 30 260 280 ratio should be around 1 2 1 for pure RNA e A gt 30 260 280 ratio should be around 1 1 8 1 for pure DNA 9 Drop Technology Version 1 3 1 0 Introduction The A260 A280 ratio is commonly used to detect contamination by relatively small amounts of nucleic acid DNA in protein solutions since proteins particularly aromatic amino acids absorb light strongly at 280nm The rever
49. ing of how their pipettes work before results obtained using such pipettes can be described as accurate e Understanding how a pipette works More than likely in the vast majority of applications in a modern laboratory an air displacement pipette is used An air displacement pipette is a bit like a syringe except that there is an air filled cushion between the piston and the sample The air cushion prevents the piston from coming into contact with the solutions which is good but it also puts some limitations on the pipette The volume of the air cushion is affected by temperature and pressure and volatile solvents can evaporate into it Each of these affects pipetting accuracy The barrel of an air displacement pipette is also vulnerable to contamination by the pipetted solution This can be an issue if you are working with corrosive or bio hazardous materials The drawings below show how an air displacement pipette works a b c d a The piston moves to the appropriate position when the required volume is set b The button is pressed to the first stop prior to sample aspiration The piston descends and expels a volume of air equal to that indicated on the volume setting c After immersing the tip into the liquid the button is released This creates a partial vacuum inside the tip and the pressure forces the volume of liquid into the tip d To dispense the sample the button is pressed to the first stop again The air p
50. logy com Fig 4 3 Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS Information ii Wavelengaa of Absorbance AU 260 230 260 280 280 Ratios Sample Type MAA Concentration ng ul dsDNA 7 ae aa eS Sample Name No 1 x Baseline Correction at 320nm 65 000 5 58 500 1 52 000 1 45 500 1 a 39 000 1 a 32 500 26 000 l 19 500 4 Measure 13 000 280 300 320 Wavelength nm TLDAson f Sample Type BSA Concentration mg ml p d Sample Name KO No 1 xB e Correction at 340nm 65 000 58 500 Blank 52 000 45 500 2 39 000 rd 32 500 26 000 19 500 Measure 13 000 6 500 o TT 200 220 240 260 280 300 320 Wavelength nm NOTE Laboratory gloves must be worn at this point STEP 1 In the TLDAsopte open the Settings tab Fill in or change the available fields highlighted opposite Integration Time Average Boxcar Sample Volume Sample Folder STEP 2 Go the relevant measurement tab Fill the required fields for measurement DNA RNA eSample Name e Sample type and factor if it is required e Arbitrary wavelength at which absorbance can be found if required e Baseline Correction deselect if you don t want it enabled Protein eSample Name eSample Type and Factor s if required eBaseline Correction deselect if you don t want it enabled eArbitrary Wavelength at which absorbance can be found if require
51. mg ml BSA 1 000 ng ul dsDNA 8 mg ml BSA SD 0 4mg ml CV 4 9 400 ng ul DNA SD 10ng ul CV 3 9 MEASUREMENT SAMPLE LOADING AND CLEANING TIME lt 10 seconds DIMENSIONS WEIGHT SAMPLE PEDESTAL MATERIAL OPERATING VOLTAGE FIBRE OPTIC CONNECTION TYPE D 454mm 17 9 X 250mm 9 9 X H 330mm 13 11Kg Quartz UNIVERSAL INPUT 100 TO 220 VAC 50 60 HZ OUTPUT 12VDC 19VDC 3AMP 3 5AMP MAX SMA 905 61 Drop Technology Version 1 3 6 0 Appendix Air bubble interference If air bubbles are contained within the sample or blank drop during measurement this will cause the light to scatter and will adversely affect the result Particular attention should be paid to samples containing high protein concentrations these samples are prone to the formation of micro bubbles Detergents are also noted to cause bubble foam formation If high concentration protein measurements or samples containing detergent are to be analysed replicate readings are recommended to offset and identify anomalous results due to these phenomena Hydrophobic sample surface The quartz sample loading surface on the plinth is designed to be hydrophobic so that the sample drop sits up proud from the quartz surface and forms a sessile drop prior to measurement Due to carryover adherence or drying of the sample on the quartz surface this property of the loading surface can be temporarily lost This leads the quartz surface to act i
52. n a hydrophilic manner which can produce poorly formed drops see Fig 3 4 b These phenomena can be exacerbated after working with high concentration protein samples due to their adherent nature The hydrophobic properties can be restored easily if the cleaning procedure is performed Section 4 1 Version 1 3 Drop Technology 62 6 0 Appendix 6 4 Parts that Require Re order Part Description Part Number Replacement Plinth Package DTO600 100205 Plinth Removal Tool DT0900 200105 Fibre optic cable DT0800 300101 Reference Standard Kit containing Drop Technology RM1 Drop Technology RM2 DT0600 100206 Blank Drop Technology RM1 DT0810 300103 Drop Technology RM2 DT0810 300104 Drop Technology Blank Solution DT0810 300102 Cleaning Kit containing 2x 50ml cleaning solutions DTOGO0 100204 Cleaning cloths Cleaning tool 63 Drop Technology Version 1 3 6 0 Appendix 6 5 Pipetting Technique Accurate pipetting is of critical importance in all areas of science this is also the case when using TLDA instruments What follows are a number of tips that will improve TLDA results and generally should be adopted in experiments Without accurate pipetting experiments would be non reproducible stock solutions would be inaccurate and assays would have such large errors that comparisons would be meaningless The accuracy of pipettes depends on their operator An operator needs to practice good technique and have a thorough understand
53. n work E DNA_with_BSA1 dsg 62KB DSG File TODT mie fe e Name dr This is the data from the dark current Bsa wth DAL peg 62KB PSG Fil measurement this is automatically obtained by the software prior to testing When exported and opened in excel column A is the wavelength and column B is the respective intensity dkd for dna work and dkp for protein work e Name dna or Name prt This file is the absorbance of the sample When exported and opened in excel Column A lists the wavelength in increasing increments and Column B lists the corresponding Absorbance at that wavelength dna for dna work and prt for protein work e Name sq This file is the intensity of the sample When exported and opened in excel Column A lists the wavelength in increasing increments and Column B lists the corresponding Intensity at that wavelength dsg for dna work and psg for protein work Version 1 3 Drop Technology 30 3 0 Software 3 2 Calibration Tab This tab is used for Calibration of the TLDAocean It acts to ensure that the instrument is performing optimally Two levels of verification can be preformed within this tab to give the user full confidence in all results that are attained The Reference Standard kit that is provided with the TLDAocean instrument contains the solutions that are to be used as the standards of calibration Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS 5 Pt Performance
54. nfigured wavelengths Graphical representation of each drop s spectrum is also displayed on the screen The spectrum covers the 200nm 750nm range Version 1 3 Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS 5 Pt Performance Check 10 Pt Validation 0 70927 0 69119 0 68456 0 69631 0 54066 0 52688 0 51807 0 52343 re 0 55836 0 55224 0 56584 0 55981 a a X Information 5 TLDAson Sample Name RM 1 01109 0 909985 0 808875 41 at 0 707766 w 0 606656 4 F S l 5 0 505547 2 Ast i 0 404438 amp 0 303328 0 202219 0 101109 o Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS Information 5 Pt Performance Check 10 Pt Validation Lambda 1 0 47863 0 43465 0 43316 0 44322 0 47176 500 x Baseline Correction at 700nm Wavelength nm ABSORBANCE Lambda 2 0 46591 0 45652 0 46047 0 47363 0 47717 Lambda 3 0 45337 0 46165 0 43568 0 50410 0 47210 Sample Name RM Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS Information 5 Pt Performance Check 10 Pt Validation Replicate Lambda 1 0 47863 0 43465 0 43316 0 44322 0 47176 0 96960 0 89661 0 93382 0 94851 0 94538 Lambda 2 0 46591 0 45652 0 46047 0 47363 0 47717 0 93348 0 92338 0 95167 0 94839
55. ngs Calibration DNA RNA Proteins UV VIS Information TLDAson A280 BCA Bradford Lowry Biuret Sample Type BSA Wavelength nm Absorbance AU 250 A280 icm path 260 280 Concentration mg ml Sample Name 65 000 58 500 52 000 45 500 Zz 39 000 32 500 26 000 19 500 13 000 6 500 o 200 Baseline Correction No 1 x Baseline Correction at 340nm Decline Meqeure 220 240 260 280 300 320 340 360 Wavelength nm For protein quantification the TLDAocean system automatically incorporates a Baseline Correction this normalises the data at 340nm i e the absorbance value at Baseline Correction at 240nm 340nm is deducted from all absorbance Sample Name Sample Name BSA10_1 Version 1 3 values at all wavelengths This is the data plotted to the screen and displayed in the necessary information boxes This can be disabled so that the raw absorbance data is recorded An appropriate sample name ought to be inputted here Drop Technology 40 3 0 Software Sample Type sample Type Other Protein tE amp Wii Wavelength Wavelength nm Absorbance AL 260 A280 41 r A280 icm path The user must select the type of Protein being measured The options that appear in the drop down list are e BSA e l gG e Lysozyme e Other Protein Emolar amp MW e Other Protein E 1 In the cases of Other Protein the user needs
56. om the liquid touching against the edge of the reservoir to remove excess liquid Dispense the liquid onto the plinth by gently pressing the operating button to the first stop This volume is equal to the set volume Hold the button in this position The liquid that remains in the tip should not be included in the delivery The remaining liquid should now be discarded with the tip or delivered back into the reservoir Release the operating button to the ready position Suitable for High viscosity Ready Position l 2 3 or foaming liquids protein and highly First stop concentrated nucleic acid solutions Second Stop Pipette slowly to avoid bubble formation Version 1 3 Drop Technology 66 6 0 Appendix Never e Put pipettes on their side with liquid inside the tip The liquid might get into the pipette barrel and cause some serious corrosion damage e Set the dial past the stated upper limit of the pipette on variable volume pipettes as this could offset the calibration e Use more pressure than is needed on the plunger as this could damage the piston over time Always e Take the ambient temperature into account Pipettes are calibrated at room temperature When working at a different temperature e g in a cold room pipettes will not dispense the displayed volumes e Take the sample temperature into account In a recent Nature Methods publication Millet and Barthlen observed a strange phenomenon where when r
57. on between the application software and the spectrometer is properly established TLDAsom is ready to be activated Currently the user can measure data read the information of the help system and get familiar with TLDAsoft but no absorbance data can be acquired because the Blank button is disabled 16 To enable all functions of the software you have to contact Drop Technology for the software registration key You should start the software and go to the information tab and note the serial number of your spectrometer This serial number may also be visible as a sticker on your spectrometer Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS Information Device Linked Identification Refresh USB4000 Yes USB4H05663 USB Key No N A Now Administrator Name Password TLDAsoft Copyright Drop Technology Beta Version 4 0 0 Drop Technology www droptechnology com 17 Register your product by emailing the serial number of the spectrometer to Drop Technology They will return by email a 30 digit Registration key and administration name and password 18 Return to the information tab of TLDAsot and once you enter in the supplied Administrator Name and Password the box for the registration Code becomes accessible gt Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS Information Device Linked Identification Refresh USB4000 Yes USB4H05663 USB Key No N A Now Administr
58. ot directory with the date and time recorded if no folder is specified This provides information for the user in regards to all functions in the settings tab This button resets the TLDAoccean system to its default settings for the sample volume integration time average and boxcar width This is the drop size in microlitres The up and down arrows can be used to adjust the Volume The Volume is set to 3uL by default NOTE Ensure that the value shown agrees with the drop size set on the pipette The integration time represents in milliseconds the amount of time for which the detector sums up the incoming photons A larger integration time results in higher peaks in the graphical spectrum because more light is detected so a greater intensity of light is recorded If saturation is a problem a smaller integration time should be used to lessen the effect that the high Drop Technology 28 3 0 Software Average Average 10 Boxcar Width Boxcar Width 10 Shutdown File Manager 29 of i _ r intensity of the light source is having on readings The integration time is set to a default of 10 milliseconds The Average field represents the number of discrete spectral results that are collected by the detector before it takes an average spectrum The average spectrum is the only one sent to the software and stored Discrete traces are not stored in the software A larger Average n
59. r suspensions in a solution without the need for a cuvette or a large amount of sample It measures the intensity of a light beam after it is transmitted through and emerges from sample drops and compares this intensity to a reference measurement to create an absorbance value by employing the standard absorbance formula the sample IBlank Incident Light I light entering A b sO rb ance Lo g 7 blank sample Where lblank the intensity of the light detected after being transmitted through the blank sample a lsampie the Intensity of the light detected after Sample ight leaving being transmitted through the sample the sample The amount of light absorbed is directly proportional to the concentration of the absorbing solutes or particles in that sample relative to the blank Version 1 3 Drop Technology 6 1 0 Introduction 1 5 4 Beer s Law The Beer Lambert Law Beer s law is employed to calculate the concentration of a sample This equation correlates the known absorbance and the concentration of the sample It takes into account the extinction coefficient of the sample tested and the pathlength of light through the sample Hence a quantitative measurement can be obtained from a spectrophotometric system A CL Where A Absorbance AU Absorbance Units e the molar extinction coefficient L mol cm C the concentration mol L L the pathlength cm This is usually represented as 1cm
60. r device Before clicking Next make sure that your device is connected to the system oe 7 If your spectrometer from Ocean Optics is not listed as shown in the example below although it is connected to one of the USB ports of the PC you should disconnect it and wait for about a minute When this time is elapsed reconnect the spectrometer to the same USB port as previously and wait again for about one minute If the device from Ocean Optics is still not visible in the list below there must be something wrong with the USB port or the spectrometer Please try a different USB port proceeding in the same manner as explained above Please contact customer service if the problem persists otherwise click on the Ocean Optics device in the list before you click Next E libusb win32 Inf Wizard ol ese Device Selection Select your device from the list of detected devices below If your device isn t listed then either connect it or click Next and enter your device description manually Vendor ID ProductID Description 0x2457 0x 1022 Ocean Optics USB4000 Ox 1BCF Ox0007 USB Optical Mouse 0x0S0C 0x 1000 Intenso Business Line 0x046D OxC312 USB Multimedia Keyboard 0x0000 0x0000 xHCI Root Hub 0 4 ur p a Core 8 The Manufacturer Name in the window below can be changed optional before pressing Next 2 libusb win32 Inf Wizard o ae Device Configuration Vendor ID hex format 0x2457 Product ID hex format
61. r used to tighten the fibre connection Part Number DTO700 400105 Fig 1 5 e Fig 1 5 e Specially designed tool for Removing and replacing the Plinth Part Number DTO900 200105 Fig 1 5 f Fig 1 5 f Cleaning Cloths in an assortment of colours Part Number DTO900 200121 Fig 1 6 Fig 1 6 Lint free tissue Part Number DT0820 300110 Fig 1 7 Fig 1 7 Workstation Optional Part Number DTO900 200115 Version1 3 Drop Technology i o SCS 1 0 Introduction 1 3 TLDAocean Inspection and Environment Before beginning any installation or operation of the TLDAocean accessory the user must ensure that all the parts are suitable for use Nothing should be visibly cracked broken or scratched Packaging and seals should be intact and unopened Inspect the TLDAocean itself for any visible faults i e the unit should not be marked or dented If there are any manufacturer faults evident or any damage has arisen from transportation contact your supplier immediately Remove the TLDAoccean from its packaging and stand it on a rigid flat surface and check that it is fully stable in its place The Drop Technology TLDAocean is developed for indoor use only in laboratories in which there is stable environmental conditions The ambient temperature should be between 10 C and 35 C and the humidity should be between 8 and 80 It is important that there is no visible condensation on the instrument If the Drop T
62. ration the protection provided may be impaired and accessory warranty may be withdrawn 1 5 Theory 1 5 1 Background Subtraction For the Nucleic acid module in the TLDA of2 software a background reading at 320nm is deducted from the absorbance to reduce noise on the baseline This 320nm background subtraction is used because spectra may be offset from the baseline during readings When this is the case the calculated nucleic acid readings may be higher than their true values It is a standard practice to have a background subtraction in these types of measurements A similar background subtraction at 340nm is performed for protein measurements These features are enabled by default in both cases in the software but can be disabled if required 1 5 2 Blank measurement In order to use the TLDAocean instrument we must first zero the device As with any spectrophotometer this is done by measuring a reference blank solution This solution contains everything except the compound of interest which absorbs light Thus by zeroing the machine using the Reference any measured absorbance is only due to the presence of the solute or suspension of interest When the reference measurement is taken it is retained in the memory of the TLDA o te Once the reference is attained the sample can be loaded and measured 1 5 3 Principal of measurement on the TLDAocean The TLDAocean instrument measures the amount of light that is absorbed by solutes o
63. rbance value at the wavelength selected is deducted x Baseline Correction nm at 630 B from all absorbance values at all wavelengths This is the data plotted to the screen and displayed in the necessary information boxes If selected the user must set the wavelength at which they want the Correction to be taken By default this value is set to 630nm If No Baseline Correction is required the box may be deselected and the wavelength field will be inactivated In this case the raw absorbance data is recorded 43 DropTechnology Version13 3 0 Software Wavelength and Absorbance The user can select the wavelength s at which they want the absorbance to be displayed The wavelength can be selected by using the up down arrows to the right of the wavelength box or clicking in the wavelength Wavelength1 nm Wavelength2 nm Wavelength3 nm 230 F 420 E 650 box and manually entering in the value Absorbancel AU Absorbance2 AU Absorbance3 AU The absorbance value displayed will correspond to the wavelength selected equivalent to a 1cm pathlength The user selected wavelength and absorbance value are not used in any calculations By default these wavelengths are set to 230nm 420nm and 650nm Maximum Absorbance The TLDAsopte interface automatically displays the wavelength and absorbance values at which the sample had the greatest MAK AL Di absorbance After measurement th
64. reen and indicates that TLDAsot can now be started Please click OK to shut the small window Driver Install Complete mses Installation successful You have now installed the TLDAsop package but without full functionality If the software does not start properly refer to the Troubleshooting section for possible solutions 13 The Windows start menu should now include the folder tlda32_ 64 In the folder you will find two entries TLDAsoft Uninstall among a few other menu items P New Office Document T Graphicode GY Video Related Programs EB adobe Reader 6 A mecad m Java Web Start Auto ue Desktop Version d amp USBdeview32 Seren fla HP Photosmart Premier m SolidWorks 2008 gt E usBdeviews e amp HP Solution Center 3 BitZipper E TMconver t Ga nn BitZipper a 3 Photosmart MSWS 7 i a gt m HamamatsuMinispectrometer y T ministrative Tools a aii ai f PASC UCUIND fm Broadcom p Cognex Be 7 2i 7 Games gt 7 A National Instruments x m Ms Workstation I HP Cool Tools S Em Office Out A Intel R Matrix Storage Manager gt a METERED ni O E Intervideo winovo BD Defauk Ey Notepad Va LightScribe Direct Disc Labeling fAtlobe photoshop 70 1 amp Microsoft office gt a Adobe TREINAR w Microsoft Office Worl F PDF Complete IM honestech VHS to DVD 2 5 SE Roxio Im Windows Media C TLDAsoft ner Ulead Photo Explorer 8 5 S
65. ressure increases inside the shaft and the tip The compressed air pushes the liquid out of the tip To empty the tip completely the button is pressed to the second stop blow out e Consider another pipette type depending on your application The information in this section relates to air displacement pipettes but in certain situations a positive displacement pipette may be a better option Positive displacement pipettes also Version 1 3 Drop Technology 64 6 0 Appendix work like a syringe but unlike air displacement pipettes they don t have an air cushion This makes them more accurate for pipetting volatile solvents and more suitable for pipetting corrosives and bio hazardous material They are expensive because the barrel is replaced as part of the tip but they can be a good option in some cases A cheaper alternative is to use and air displacement pipette with barrier tips however these only address some of the issues The below picture shows 2 positive displacement tips plungers Practice Good Pipetting Technique Know how to pipette properly Pipette with a slow smooth action Hold the pipette vertically when drawing liquid in Only immerse the tip slightly when drawing liquid in otherwise the outside of the tip will be coated with liquid which will be transferred along with the volume inside the pipette When dispensing the liquid hold the pipette vertically but keep the sidewall of the receiving vess
66. ry concentrated gt 2 000ng ul a volume of 1 0ul could be loaded to reduce error 13 Drop Technology Version 1 3 2 0 Installation STEP 1 Screw one end of the fibre optic cable onto the SMA905 connector on the lid of the TLDAocean Once finger tight use the 8mm spanner to tighten another 30 The other end of this fibre connects to the light source used Fig 2 1 TECHNSLOGY tn TULDA Nsm er Mitteg Light Drop Analy NOTE The fibre must not be loose STEP 2 Using a screwdriver unscrew the clamp at the rear of the TLDAocean Fig 2 2 Fig 2 3 STEP 3 Fit the clamp around the fibre optic cable and screw it back into place Fig 2 3 This retention clamp is designed to minimise any risk of damage to the fibre STEP 4 Place the TLDAocean along with the light source and spectrometer on the workstation Fig 2 4 a Version 1 3 Drop Technology 14 2 0 Installation Fig 2 4 b STEP 5 Ooi Screw the other end of the fibre optic USB4000 cable from the top of the TLDA to the light ye source Screw one end of the shorter fibre optic cable to the SMA 905 connector on the bottom of the TLDAocean and the other end to the spectrometer Fig 2 4 b eS ee STEP 6 Make sure the light source is switched on and that it is connected to the spectrometer if pulsed light is required Connect the USB B connector to the spectrometer and the USB A connector to the computer
67. se however is not true it takes a relatively large amount of protein contamination to significantly affect the A260 A2289 ratio of a nucleic acid solution Typical A260 Azgo ratio values for protein contaminated nucleic acid and nucleic acid contaminated protein samples can be found in the tables below Table 1 Protein vs Nucleic Acid Table 2 Nucleic Acid Vs Protein ratio table ratio table Protein Nucleic A260 A280 Nucleic Protein A260 A280 10 o0 057 0 10 acid 132 1 73 A260 A280 ratio has a high A260 A280 ratio lacks sensitivity for Nucleic acid sensitivity for Protein contamination in Protein contamination in Nucleic Acids This difference exists because at 260nm and 280nm nucleic acids have a much higher extinction coefficient than protein For this reason relatively high concentration of protein will contribute relatively little to the absorbance at 260nm and 280nm i e the nucleic acid will dominate results While the protein contamination cannot be reliably assessed with an A260 Azgo ratio this also means that it rarely contributes to error in the quantitation of nucleic acids 8 65319 7 78787 6 92255 6 05723 5 19192 4 3266 Fig 2 Screenshot of nucleic acid sample with Protein contamination at 280nm 3 46128 2 59596 10mm Absorbance AU 1 73064 0 565319 0 T PL sed L a a LUS L a L imac ml ea A 200 220 240 260 280 300 320 340 360 380 400 Wavelength nm Th
68. to input the respective values prior to taking the measurements For Other Protein Emoiar amp MW the user must input a Molar Extinction Coefficient Emolar and a Molecular Weight MW for that Protein sample For Other Protein E 1 user must input a value for the Mass Extinction coefficient based on a 10mg mL sample For BSA IgG and Lysozyme the mass extinction coefficients are preconfigured See Section 1 6 Theory If the absorbance at a particular wavelength needs to be known it can be selected here The wavelength can be selected by using the up down arrows to the right of the wavelength box or clicking in the wavelength box and manually entering in the value The absorbance value displayed will correspond to the wavelength selected The user selected wavelength and absorbance value are not used in any calculations Proteins absorb significantly at 280nm so the absorbance at a 280nm is always measured and displayed here This absorbance values displayed are equivalent to a sample with a 1 cm pathlength Drop Technology Version 1 3 Ratio 260 280 Concentration Concentration mg ml Graph 27 2128 24 4915 21 7702 19 049 16 3277 m w fmi D T t m Absorbance AU Oo lt 10 9851 8 16384 5 44256 2 72128 D Version 1 3 240 260 280 300 320 Wavelength nm 340 360 380 400 3 0 Software The A260 A2g0 is a ratio of the absorbance values
69. trations are calculated using the mass extinction coefficient of 13 7 at 280nm for a 1 10mg ml IgG solution e Lysozyme Unknown Lysozyme protein concentrations are calculated using the mass extinction coefficient of 26 4 at 280nm for a 1 10mg ml Lysozyme solution It also has 2 user definable molar and mass extinction coefficient options e Other Protein Emolar amp MW User defined values for molar extinction coefficient M cm and molecular weight MW in KiloDaltons kDa for their respective protein reference The Emolar and M W should be entered prior to making measurements e Other Protein E 1 User defined values for mass extinction coefficient Lgm cm for a 10mg ml 1 solution of the respective protein reference The appropriate extinction coefficient should be entered prior to making measurements NOTE Due to the patented design and principal of operation of the TLDAocean optics the unique sampling method ensures exceptional accuracy and reproducibility of results NOTE It also means that the volume loaded dictates the pathlength through the sample In order to get the maximum accuracy from these optics it should therefore be understood that lower volumes confer more accuracy at higher concentrations and conversely higher volumes confer more accuracy at lower concentrations e g For nucleic acids if the sample is very dilute lt 100 ng ul a volume of 3ul could be loaded and inversely if the sample is ve
70. umber will reduce noise as the Signal Noise ratio improves by the square root of the number of Averages However resolution decreases as the Average number increases The average number is set to a default of 5 The Boxcar Width represents the number of pixels that are averaged together to give a smoother spectral acquisition The number shown represents that number of pixels to the left and that number of pixels to the right averaged together Boxcar width is applied to the acquisitions after they have been averaged Increasing this may give a smoother plot but decreases the resolution of the plot The Boxcar width is set to a default of 5 This shuts down the TLDAsore program This must be done before the TLDAocean is switched off File Manager opens up the typical Windows Explorer window that contains all the data files From this window files can be copied deleted or opened from the PC s memory They can also be transferred to a USB for exporting Drop Technology Version 1 3 3 0 Software Files Types For any particular sample there are four data files saved with that name The data files are distinguished by their extension nabs in Extensions are Se oe he e Name bk This is the data acquired from the r eg blank measure When exported and opened in a se an excel column A is the wavelength and column B alae ee ae is the respective intensity 76 ae bkd for dna work and bkp for protei
71. upe ates PW OM RS cto deca gteresia sates decease ss dence aeeacteteene ane aiden pantera eee 2 1 3 TLDAGeegn INSPECTION ANA ENVIFONMENE wcssccsiaceiicssisivcdein eb ddead n 5 TA Sarre UW NOCO rth ica an din stirs male neutanuaitalaann iawen moet an AT 5 MES TSS Oates re tc es ti a atte latin acs a it ele ace A wind easter ance ena le 6 ZO VST AN ACI ON i E E ANR 14 2A TELDA ocean ASSE MDI Y a E E 14 2 2 SOtWare Tnstallai O Meena E E EEEE EAEE E E 16 3O ON EO a R E T E 25 3 Basic FUNCHON BUNONS icisiciacotin ia a A E a NE 25 ILE a Da eo ne Ree UP RET tc tona Sa E E AA 27 F UNO UO O trays tae EAA E TE AOAN A O 31 So ONARNA TaD e E E A E ee eee ee ere eer 38 SA Protem T iDan a a ed azcateuat E dapetenianta eh E A EAN 40 IS UVANI CAD aen E T T E E T 43 SOs TON Ul Ol aoan e A R 45 SY aes 0 B62 6 UDE IAE an pene ne a eR rR RE I ten TOA 47 40 General ODEl AU OM iesen neninn Eaei 48 BA EDO AG ae sa ANCONA EY esa E N E E TN 48 42 SAMMI Evapora tO Mear aesnenied sauna a 48 AS FARING aie aSUre Ment caivrastucctiaisain eh Atecatatontoidusiwasanh Adecataiemaadatiiaen att 49 5 0 IVIAINUS NANG C ies sssinscrnshcntacnmaatumanndstwdietiednsesscdsienesesadsionsanbadonsinnes 55 Dl Cla MS the IU ee E a 55 5 2 Method of Cleaning the Phthiria a n 55 DS REDIAGING the oP MMU oasen e e li uaautdeatiei mmm diiadie Sanat deans 56 5 4 Method Of Replacing the PHU scxvsusitessic
72. will be enabled 8 16384 5 44256 2 72128 The result values will populate in their designated boxes By pressing Accept the data for the absorbance spectrum will be saved to the memory of the PC Fig 4 15 STEP 12 Once this measurement is completed open the sample chamber If desired the sample can be retrieved using the Drop Technology Pipette EN KIMTECHE TIT Alternatively the sample area can be cleaned using lint free tissues Fig 4 15 Lint free tissues are recommended by Drop Technology for their highly absorbent properties and because the presence of lint could affect the absorbance result NOTE The sample plinth must be cleaned properly using these wipes between each sample 53 Drop Technology Version 1 3 4 0 General Operation STEP 13 The procedure is then repeated for the subsequent sample drops The absorbance values will be displayed at the selected wavelengths Version 1 3 Drop Technology 54 5 0 Maintenance e Though an absorbent lint free tissue used between samples removes sample carry over decontamination of the plinth surface should be carried out regularly e If a sample is i high in concentration ii biological iii a protein or iv a large amount of samples are tested the plinth should be cleaned between each set of tests with the Drop Technology Cleaning Fluid The cleaning interval is best determined according to the conditions of use
73. wn colour coded and overlayed on the screen After ten readings have been accepted TLDAsore Will automatically calculate the mean and standard deviation for each wavelength which is displayed at the bottom of the table of data Once you double click in the table of data the data table will fill the screen TLDA sone Will state whether the TLDAocean instrument has passed or failed based on the reference standard specifications The test should be repeated as above with the second reference fluid You will have to select the second reference from the drop down menu reblank the system and measure ten drop samples The data will be displayed on the screen and a pass or fail status displayed In the event of failure see Section 6 3 Troubleshooting Version 1 3 3 0 Software 3 3 DNA RNA tab To measure nucleic acid samples select the DNA RNA application tab These samples can be easily checked for concentration and purity using the TLDAocean spectrophotometer Transmitted Light Drop Analyser Settings Calibration DNA RNA Proteins UV VIS Information i TLD As n S i oo Wavelength nm Absorbance AU 260 230 260 280 280 Ratios v Sample Type Concentration ng ul dsDNA x Sample Name ni x Baseline Correction at 320nm 65 000 58 500 52 000 45 500 a 39 000 32 500 Decline 2 26 000 19 500 13 000 Measte 6 500 l 0 200 220 240 260 280 300 320 340 360 380 Wavelength
74. x 353 0 1 452 3967 info droptechnology com www droptechnology com

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