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DropSense96 User manual

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1. 63 5 2 3 DropPlate96 sample TaVOUt saei M ca oM duplo NUN aes 68 5 2 4 Extra Measuring multiple proteins with different extinction coefficients 68 5 2 5 Extra quantification of fluorescent labeled proteins eese nennen 71 5 2 6 SEGIEIMCOSUIOCIN cag mcr 73 5 2 7 Dara send eae 74 5 2 7 1 ise bosco a abut Copt 75 5 2 7 2 pay 76 5 2 7 3 77 5 2 7 4 CES play 81 5 3 GENERAL UN SPECTIROPHOTOMETRY E 83 5 3 1 ON TU 83 5 3 2 medsuremelE desi o US ERR T A putt qM eee 83 5 3 3 DropPlate96 Sample diss 87 5 3 4 Start Measurement cohen apes Ev Mai co i petas rtm td nc 88 5 3 5 aes 89 5 3 5 1 Sewell diSpldy v iHa o dc ves vc rua E Levi 90 5 3 5 2 Cola E SICHERER DECLA AEE CS DU VE 91
2. 230 10 16 25 A260 10mm 30 96 2 0 LOmm 15 32 Raw A405 10mm 0 27 260 230 191 2160 280 2 02 Change settings blank B subtract background correction default 405 IL cingle point Export of spectra and Change grafic X and Y scale sample data to excel Extra tools are included for detailed analysis and comparison of the samples e Hide Show cursor with this cursor a specific wavelength can be selected by dragging the blue cursor The selected wavelength and its OD value 10 mm is displayed Page 56 e Path length selection Above the spectral view a choice can be made between OD 10mm or OD 1 0 mm OD 0 2mm for a DropPlate D measurement or OD 0 5mm for a DropPlate S measurement Note that the Y axis scale will automatically adjust to the proper OD values When selecting OD 1 0 mm OD 0 2mm the 1 0mm OD spectra will be shown with a full line while the 0 2mm OD spectra will be displayed using a dotted line e Overlay spectra when pressing the CTRL button on the computer keyboard select two or more samples from the select input well table to overlay the selected spectra When using the shift button and selecting two samples all samples between the selected will also be displayed on the screen Underneath RNA samples are shown Results e trinean Results 2 REPORT DISPLAY SINGLE WELL DISPLAY COLUMN DISPLAY 96 WELL DISPLAY
3. Continu to the Plate layout screen When entering the blank and sample positions manually on the plate layout screen proceed as described in section 5 1 3 and press then press the Enter check E1 button Now a table will appear with a overview of the selected wells Type in the proper E1 for each protein or copy past them from another table All the information can be saved in excel by pressing the export excel button at the bottom of the screen Press Save to return to the Plate layout screen Manual blank and sample positioning i inean Plateg_ayout Bel O00000000 meoooo9o009 555000 un El mu LELEL IMP OF n cumin ied edad PLATE LAYOUT PLATE LA Open extinction coefficient overview Page 69 When using the import option section 4 1 4 2 an extra column selection can be done so the software can import all E1 extinction coefficients for the protein samples Upon returning to the plate layout screen an overview of these E1 coefficients can be shown by clicking on the enter check E1 button When using the Replicates function see section 4 1 4 1 entering the extinction coefficient of one protein sample will automatically lead to the fill in of the extinction coefficient of its replicates Import Samples
4. Pump Concantration ASEO A230 ATZGQ AZBO Ing ul bark 1 0 00 3 diDMA 1 256 66 239 dsDNA i LI Create report 55 43 2 1 83 diDMA 27 81 130 aD 13 37 i 191 diDMA 27 2 3 158 diDMA EXPORT RESULTS _save_ save asf rename Save as format Page 61 5 2 Protein quantification 5 2 1 Theory Protein concentration and purity determination Protein concentration like nucleic acids can be determined by measuring their UV absorbance at 280nm and calculating the concentration using the extinction coefficient of the protein in the Beer Lambert equation This method is suited to quantify purified proteins However the 280nm absorption of proteins depends on the presence of aromatic amino acids Trp Tyr and Phe and Cys Cys disulfide bonds Therefore the UV absorption of proteins varies greatly and depends on the particular amino acid concentration of a protein In addition buffer type ionic strength and pH affect the UV absorption and even pure protein solutions may have different conformations and modifications When performing protein A280 concentration measurements the best approach is to empirically derive the extinction coefficient for the protein of interest or search for published protein extinction coefficients examples in the Practical Handbook of Biochemistry and Molecular Biology Alternatively if the p
5. 5 5 aa Once the full plate layout has been completed this information be saved for future use The export plate layout button at the bottom opens a pop up window offering the choice of several formats txt xls csv to save the information When selecting Excel an 96well matrix overview is made in the sheet This can be useful in data analysis or reports in the experimental log book bimk 17 bnc 25 benc ar bnc 49 bene s 2 10 sample 18 sample 26 semple 34 sample_42 sample_s0 samples o C semple 3 19 sample 27 semple 35 sample_43 sample_st samples9 D sample 4 sample_12 sample_20 sample 28 semple 36 sample_a sample 52 sample_60 o E semple 5 sample_13 sample_21 sample 29 sample_37 sample 45 sampe 53 sempe J o F sample_6 sample_14 sample 22 sample_30 semple 39 sample_46 5 sample amp 2 6 7 sample_15 sample 23 sample 31 semple 39 sample_47 sempe 55 sample amp 8 as ooo o ooo ooo o 34 4 1 4 2 Import of plate layout by file CSV XLS and TXT import An import function Import Plate layout is foreseen at the bottom of the Plate Layout screen for fast and easy entering of plate layout information Several types of files
6. Single well display Gives a detailed view of a sample spectrum and all additional sample data next to the spectrum A sample can be viewed by clicking on the name of the well or sample in the select input well table or by clicking on a well in the 96well overview Results ae Results REPORT DISPLAY Select InputWell Choice of spectra shown on display COLUMN DISPLAY V SINGLE WELL DISPLAY hide cursor OD 10mm A2 sample 9 autoscale Selected well B2 sample 10 for display a sample 11 D2 sample 12 E2 sample 13 F2 sample 14 G2 sample 15 H2 sample 16 blank 17 B3 sample 18 sample 19 sample 20 sample 21 sample 22 96well E oveview BACH TO DATABASE Show hide cursor with display of wavelenght and OD E wavelength nm 280 lomm Absorbance OD 139 Concentration mg ml 0 57 mg m Position C2 Sam ple Type sample information Blank Al Name sample_ 41 al 24 30 Concentration 0 57 mg ml 260 280 0 56 Change settings blank 340 350 380 fd subtract Wavelength QUICK EXPORT TO XLS EXPORT B background correction default single point 405 nm Export of spectra and Change grafic X and Y scale sample data to excel Extra tools are included for detailed analysis and comparison of the samples e Hide Show cursor with this cursor a specific wavelength can be selected by dragging the blue cursor The s
7. For every selection the sample properties used for the concentration calculation are shown on the right side Page 42 v dsDNA ssDNA RNA Oligo Choice nucleic acid sample Sample properties Unique features for measurement of oligo nucleotides are included in the software Different oligo s can be measured in the same experiment For each oligo the molecular weight extinction coefficient and concentration factor is measured see 5 1 4 Solvent Select the solvent used for the samples to be measured This solvent information is used in the DropQuant software of the DropSense96 to fine tune the pressures used by the vacuum pumps inside the instrument guaranteeing precise and controlled filling of the microfluidic structures All water based solvents containing low amounts of salts and buffers are grouped in one option since these substances don t affect the pumping properties v water based solvents incl TE 5 RT buffer water glycerol 5 10 water DMSO 5 10 Choice solvent Extra button for high viscous samples Page 43 Field tests have shown that genomic DNA lambda DNA and highly concentrated nucleic acid samples can be very viscous and non homogeneous This can hamper both precise pipetting of the DNA samples as well as precise transport of the sample in the microfluidic DropPlate An extra button for high viscous samples can be used so the pumps will work at higher pressure and longer
8. Note that the values reported are NOT nomalized to a 10mm pathlength so if comparison is being made to other systems the appropriate factor should be used to convert them x10 for 1mm pathlength x50 for 0 2mm path 5 3 2 Starting a UV Vis measurement Pressing the new measurement button on the main menu opens the following screen lt New measurement oan Start new measurement Experiment definition Experiment 2010 10 20 15 52 NudeicAdds purified Proteins General uv _ Sample definition po mu Select DropPlate type path length Y Owl ff ss Save as Format _ Task bars CANCEL OPEN TRAY NEXT gt All options on this screen are described in detail in section 4 1 2 For general UV Vis measurements select the proper preferences in the sample definition box Page 83 Sample select General UV Vis the button turn light gray upon selecting Sample material and information Additional information can be entered like a name of the solution to be analyzed An extra text box is foreseen for additional information OD at wavelength nm A selection of wavelengths can be designated in the list using the Add and Delete buttons These wavelengths will be used for OD calculations and this data will be included in the report Solvent Select the solvent used for the samples to be measured This solvent information is used by the DropSense96 to fine
9. Page 31 Blanking information It is recommended to position the blank references first then the samples ite Layout Plate Layout Blank selection poop0o0000000 EIEIE EIE E E ETE E E EJ EXE EJEJ EJ EJ EJ 8 EX E EXPORT IMPORT CANCEL OPEN TRAT PREVIOUS STAAT TEST gt SAMPLES SAMPLES A choice can be made between Autoblank the background absorbance is automatically set to zero or Blank a blanking reference included in this experiment When several blanking solutions are used and positioned on one or multiple DropPlates96 for the experiment then these different blanks can be chosen as reference for any samples on any plate of the experiment The software automatically presents the entered blank wells where the user can select from sye average of blanks DropPlate 1 1 DropPlate 1 C3 DropPlate 1 D12 Another option in the software is to take the average data of all blanks included in the experiment and use that info as blanking data for the samples Page 32 For sensitive measuring of low concentrated nucleic acid samples example dsDNA lt 100ng yl We recommend to always use a blank sample The autoblank method is a software adjustment of the spectra background correction and can be less accurate then when using a proper blank sample Sample information Select sample as Type well Change the sample name if needed and select what t
10. mg ml OD 10mm OD 10mm OD 10mm DropPlate1 A1 0 0 0 00 28 DropPlate 1 186 125mg m 1208 165 1655 004 052 DropPlate1 C1 186 O1mg m 009 012 012 002 049 DropPlate1 D1 BSA O2mg m 014 002 04 DropPlate1 E1 IgG_O 8mg ml 079 108 108 001 05 DropPlate1 F1 BSA L mg m 083 114 114 001 055 DropPlate1 G1 186 63mg m 610 837 837 0 04 05 DropPlate1 H1 BSA 125mg m 624 854 854 005 057 Page 80 5 2 7 4 Report display The report section gives an overview of the experiment information and all the measurement data in a table format The info on the data table can be altered by clicking on the header of the column concentration 260 230 or 260 280 ratios can be chosen Furthermore the measurement description can be altered by pressing the edit button This overview can be exported to several formats like a CSV txt excel sheet or pdf file The data can also be printed directly to a printer of choice Choice of data shown in the column Results Results IE ael REPORT DISPLAY SINGLE WELL DISPLAY Concentration MEASUREMENT DATE TEST PERFORMED BY Measurement quen Name Concentration info sois PATH LENGTH blank 1 0 00 mg ml Double path length Bl sample 2 1 26 mg ml i 0 76 EXPERIMENT NAME sample
11. Liquid handling robots with 1 to 96 pipetting heads can be used for dispensing samples in the DropPlates The pipetting head must approach the DropPlates vertically and the distance between the head and the input wells should be optimized to ensure that the dispensed sample can make contact with the inner wall of the input well the pipetting head shouldn t make contact with the input wells The conical shape of the input well and its hydrophilic nature will guide the droplet deeper in the input well to ensure that the sample is drawn into the anti evaporation reservoir For additional information please contact info trinean com 3 1 4 Sample properties Sample carryover is prevented by the DropPlate design the elevated edges of the input wells avoid sample carryover within a DropPlate and the extra safety features on the DropPlate avoid contamination of the pressure manifold of the DropSense96 Sample homogeneity needs to be ensured for measurement precision Sampling from non homogeneous solutions can cause significant deviations in the data generated using small volume spectrophotometers Highly concentrated nucleic acid samples and other viscous solutions are common examples known to the molecular biologist Due to the small volume requirements by the DropSense96 it is important to ensure that the sample being measured is homogeneous and not too viscous This is important for correct pipetting and avoids measurement deviation in the data g
12. Select InputWell show cursor OD 10mm sample 3 autoscale sample 4 Concentration ng ul sample_5 sample 6 882 81 ng ul sample 7 c2 sample 8 1426 04 ng ul E2 1008 52 ng ul 2 sample 9 sample 10 11 D2 F 1238 52 ng ul G2 1090 75 ng ul H2 933 32 ng ul sample 12 sample 13 sample_14 10mm Absorbance OD sample_15 sample_16 blank 260 280 300 320 340 360 380 v subtract Wavelength gt background correction EXPORT SELECTED TO XLS default single point 405 nm BACK TO DATABASE EXPORT Additional tools are included to change the analysis settings of the measurements Changes made within these tools explianed underneath stay fixed within the other data analysis screens and the export options Page 57 e Change scale Both the scales of the X axis and the Y axis can be altered for a detailed view by changing the highlighted wavelength at the beginning and end of the scale The setting autoscale has to be turned off when changing Y axis Note that the report option All absorbance values 10mm will show all the absorbance values within the selected wavelength range of the X axis So manual changing of the X axis scale will change the info in the report e Blank subtraction When using a blank reference sample it is automatically subtracted from the sample absorbance before concentration calculation This feature can be turned off so the spectr
13. absorbance at a particular wavelength the extinction coefficient concentration of nucleic acid path length of the spectrophotometer cuvette The DropSense96 performs a optical OD measurement using path lengths insured by the choice of DropPlate to enable quantification of nucleic acid samples with a wide concentration range without need for dilution The absorbance data are archived in the database and displayed on the software screen after measurement In the nucleic acid application the software chooses automatically which path length generates the best absorbance values and normalizes this measurement to a 1 0 cm 10 0 mm path These are displayed in the software for further data analysis 1 2 DNA RNA purity determination Residual cellular contaminants like proteins or compounds used in the DNA RNA preparation frequently remain present in the DNA solution and often interfere with the measurement at 260 nm leading to incorrect results Both protein and DNA absorb UV light but have different absorbance curves The peak of light absorption for DNA is at 260 nm while proteins absorb strongly at 280 nm mainly due to tryptophan and tyrosine side chains Therefore the purity of Page 40 a DNA sample can be calculated by examining the ratio of the two absorbance values A260 A280 values of 1 7 to 1 8 predict clean DNA good quality RNA will have a 260 280 ratio of 1 8 2 0 Lower values may be indicative of
14. excel txt and csv can be imported and the data can be reformatted into a dedicated DropQuant software table for quick plate layout configuration Selection of file type x Open an excel import template Import Samples Import Samples J xls xisx csv open template Import format Default converted sample s 0 more info selected sample s 0 select all deselect all Source DropFrame DropFrame Sample DropFrame Blank Position iD Position type ID of blank Position CANCEL Excel txt or comma separated values CSV file selection CSV files can be selected using the browse button and the table underneath will be filled in automatically The appropriate delimiter needs to be selected to ensure that the table is filled in properly Txt files can also be uploaded by direct selection using the browse button or by the copy drag and dropped option Like CSV files an appropriate delimiter needs to be selected Excel files can also be directly added in the import screen via the on screen browse button After importing the information from the excel file the associated table below is filled in automatically Selection of the appropriate sheet of the excel file is needed for proper table formation Page 35 Select delimiter type to create table mif 7 1 4 Open CSV file with browser button Automatic displayed table Import Samples LL BAGS example ctv ope
15. identified by a next to the label name may not be edited or deleted Page 71 Press the Save changes button and then Back to return to the New measurement page Labels Labels Database Nam Extinction Coeff Absorbance wavelength 260nm correction 280nm correction A 0 43 0 56 0 00 0 03 0 00 0 01 590 650 Alexa Fluor 660 132000 663 Cy3 5 150000 581 0 00 0 00 5 5 250000 675 0 00 0 00 3 DyLight 488 70000 493 0 23 0 15 E Add Label 0 03 0 04 0 14 0 13 Save Changes EH Page 72 5 2 6 Start measurement Press the Start Test button on the task bar of the Plate layout screen This will open the microplate tray of the connected DropSense96 and the following screen will appear 1 2 3 4 5 T 8 9 10 12 gt O 1 1 1 1 1 4 1 4 1 1 1 ABORT TEST Please insert disposable 1 DropPlate 1 in the tray and press OK to continue Cancel Load the first DropPlate DropPlate1 on the tray and press The tray will be pulled in and the measurements will start Press Cancel if the measurement should not take place The software will return to the main menu The progress of a measurement can be observed in the small measurement screen showing wells that have been measured indicated by a green color and done wells being measured orange and indicating busy and wells th
16. 20 3 1 4 SOMPE DOPET ES svn ovni E enlm d da RUE 20 3 2 DROPSENSESGIVIODE OP ACTION apodo deu seg utn enam o deg patte dere tao du uda ad dee tdeo 21 3 2 1 JUSCFIIPenTt S COTE UD s ec E E eet ena ae 21 3 2 2 Loading of a DropPlate96 on the 21 DROPQUANT SOFTWARE na utra en AR dea ete nua me Ta uM ben MEUS bue UD IUE ds 23 4 1 1 gt a D 23 4 1 2 MEO US o setae ft n uideat e 23 4 1 2 1 Drop uant MUI NUE 23 4 1 2 2 DITS TR 24 4 1 2 3 Sustemanto amd Set EITIBS 25 4 1 2 4 Measurement databases E OO HUY FIT Aen 27 4 1 3 New Imegsureliblo es o Ur dog uaa 28 4 1 4 DrobpPIgte96daVOUL Cid 30 4 1 4 1 Manual Cty OF sample IID S eae c eo op rv Ciel cepi otto de 30 4 1 4 2 Import of plate layout by file CSV XLS and TXT 35 hix e THON ecc Em 40 5 1 NUCLEIC ACID QUANTIFICATION AND PURITY
17. 3 4 02 mg ml 0 53 Experiment 2009 12 06 2 18h 08 01 sample 4 0 52 mg ml 0 65 El sample 5 3 46 mg ml 0 53 Sample F1 sample 6 1 25 mg ml 0 73 data INFORMATION Purified protein measurement BACK TO DATABASE OPEN TRAY sample 20 Export and print options 4 43 mg ml EXPORT 61 sample 7 1 21 mg ml 0 83 sample 8 1 82 mg ml 0 59 SOLVENT sample 9 1 29 mg ml 0 73 A as sample 10 1 32 mg ml 0 73 MATERIAL sample 11 1 95 mg ml 0 51 General sample 12 3 59 mg ml 0 51 sample 13 3 93 mg ml 0 53 sample 14 1 99 mg ml 0 53 sample 15 1 87 mg ml 0 58 i sample 16 1 26 mg ml 0 80 sample 17 1 95 mg ml 0 53 1 New DropPlate sample 18 1 93 mg ml 0 54 sample 19 4 37 mg ml The Export button opens a new screen to create a data report as desired In the results export sheet a selection of columns can be made to be included in all three report types The order of the columns can be altered with the move up and move down buttons A preview of the report table is displayed at the bottom of the screen The export file type can be selected by clicking on the preferred file type Furthermore the information from the summary experiment description experiment name solvent and an overview of the plate layout can also be included by activating the include summary button The report design can be saved in a format for future use by clicking the s
18. DropQuant software of the second PC o Import settings Import folder with saved formats and databases from another PC with DropQuant software 4 1 2 4 Measurement database All measurements performed are automatically stored in archive files location to be determined by the lab manager and can be opened using the measurement database button on the main menu All measurements made by a user log in for a given calendar day and time are stored in a single archive file A unique file extension drop has been given to these files to enable automatic startup with the DropSense96 software Clicking on the new search button in the task bar will open a search menu allowing searches by user experiment name keywords or date Select the measurement of interest blue highlighted and press the open data button All information of the measurements will be displayed as a novel measurement see further and the spectra are re plotted for further analysis Selected measurements can also be deleted or compressed by zipping for other use Database Database e cCrinean Date User Description 12 06 2009 Guest Experiment 2009 12 06 18h 08 Purified protein measurement 12 06 2009 Guest Experiment 2009 12 06 14h 07 Ful 96wel DNA conc measurement Search Search database CANCEL SEARCH Delete selected experiment ZIP selected experiment NEW SEARCH OPEN DATA Page 27 4 1 3 New measurement Th
19. DropSense96 manual the DropQuant installation CD Rom and a DropPlate96 frame The DropSense96 is designed only for indoor use under the following conditions e Temperature 10 35 C e Humidity 10 90 If you use the instrument in a room subjected to extremes of temperature change during the day or before use stored in a cold environment it should be allowed to come to thermal equilibrium for 2 3 hours in the laboratory before switching on This will prevent calibration or self test upon start up failure as a result of internal condensation Make sure that no DropPlate disposables are located inside the DropSense96 instrument during transportation This may cause damage to the internal mechanics of the DropSense96 Page 11 2 2 Initial start up 2 2 1 Computer requirements and software installation The operating software will only run on a PC meeting the below criteria No Mac versions of the software are currently available Microsoft Windows XP Vista or Windows operating system Microsoft NET Framework version 2 0 or higher 1 5GHz or higher processor CD ROM drive 1GB or more of RAM 2GB if running Vista 100 MB of free hard disk space for software installation Free USB port the instrument can only be connected via the USB port Microsoft Excel and Adobe pdf reader to manipulate archived data optional The system software must be loaded onto the PC before the USB cable is connected Administrator access
20. Import Samples r I E II Q import format Default header not induded Blanking information header B Source Plate iD j column 1 c DropPlate ID column DropPlate ID of blank ETT Source Postion EM I Position Blank Position EM LO Sample name 1 converted sample s li more info selected sample s 0 select all deselect all Source Source DropPlate DropPlate Sample le DropPlate Blank Plate ID Position 10 Position type ID of blank Position at T iinis spin mici CANCEL Assign a column from the data sheet that contains the extinction coefficients Page 70 5 2 5 Extra quantification of fluorescent labeled proteins The labeling efficiency of fluorescent labeled proteins used in protein arrays can be easily determined with the DropSense96 A full spectrum scan with the DropSense96 allows measurement of protein absorption at 280 nm while detecting the incorporated fluorescent dye at their absorption peak In a single mesurement the DropQuant software calculates the protein concentration is expressed in mg ml and the dye concentrations in pmol ul Continue on the Start new measurement screen Fill in this screen as described in section 4 1 2 Choose as sample type Purified proteins and choose the option L
21. Page 22 4 DropQuant software 4 1 1 Start up To ensure that the DropQuant software can communicate with the DropSense96 instrument connect the instrument to the computer and turn on the DropSense96 prior to starting the software otart the operating software by selecting the following path Start Programs DropSense 96 or by a double mouse click on the DropQuant software icon on the desktop 4 1 2 Main Menu The software opens to display the main menu 4 1 2 1 DropQuant Log in DropQuant Login Login Password connect to DropSense EXIT This screen is used to o Change the user name account o Exit the DropSense96 software o Choose to connect the software with the DropSense96 or use the software for data analysis without connecting to the instrument Pre defined accounts are o Lab manager This account has full access to the software and has the possibility of changing the number of accounts their passwords and the location of the measurement database It is recommended to change the passwords of the different accounts when installing the DropSense96 The lab manager log in is protected by a predefined password This password differs on every DropQuant installation CD and the correct password can be found on the DropQuant CD included in the installation box Page 23 o User no predefined password Second level can use the full operational software Guest no password needed L
22. analysis and comparison of the samples e Hide Show cursor with this cursor a specific wavelength can be selected by dragging the blue cursor The selected wavelength and its OD value 10 mm is displayed e Path length selection Above the spectral view a choice can be made between OD 10mm or OD 1 0 mm OD 0 2mm default for DropPlate D measurement OD Page 92 0 5mm default for a DropPlate S measurement Note that the Y axis scale will automatically adjust to the proper OD values When selecting OD 1 0 mm OD 0 2mm the 1 0mm OD spectra will be shown with a full line while the 0 2mm OD spectra will be displayed using a dotted line e Overlay spectra when pressing the CTRL button on the computer keyboard select two or more samples from the select input well table to overlay the selected spectra When using the shift button and selecting two samples all samples between the selected will also be displayed on the screen Additional tools are included to change the analysis settings of the measurements Changes made within these tools explianed underneath stay fixed within the other data analysis screens and the export options e Change scale Both the scales of the X axis and the Y axis can be altered for a detailed view by changing the highlighted wavelength at the beginning and end of the scale The setting autoscale has to be turned off when changing Y axis Note tha
23. at the bottom of every screen This allows the user to cancel the experiment open the microplate tray at any time and continu to the next software screen Within one experiment all Dropplates need to contain the same sample type as selected on the screen If the DropPlates contain different sample types than these DropPlates need to be measured as separate experiments Page 29 4 1 4 DropPlate96 layout Pressing the next button on the Start new measurement screen will open the screen where information on sample positioning on 96well display can be entered The positioning information can be done manually or can be imported from an excel text or csv file Number of DropPlates96 Plate Layout trinean Plate Layout DropPlate s Manual well definition Type well Sample et Wellname sample SS EXPORT IMPORT OPEN TRAY lt PREVIOUS START TEST PLATE LAYOUT PLATE LAYOUT Overview well names Import export functions Selected DropFrame overview 4 1 4 1 Manual entry of sample ID Defining the number of DropPlates96 The number of DropPlate96 aluminum Dropframe or a full plastic DropPlate96 to be measured can be chosen with the add DropFrame button and specific names can be given Unnecessary DropPlates96 can also be removed Furthermore a DropPlate with all the positioning information can be duplicated creating a new DropPlate with identic
24. can contain both DNA or RNA Upon returning to the plate layout screen an overview of these sequences and properties can be shown by clicking on the enter check sequences button Import Samples import Samples Is template import format Default Nrofrowsto skip header not induded Blanking information 9 header Source Plate ID J y DropPiate 10 DropPlate I0 of blant Source Postion Piesa z position Blank Position 7 Samole x press enter to confirm wea aa a converted sample s 0 more info selected sample s 0 select all deselect all Source Source DropPlate DropPlate Sample L Sample DropPlate Plate ID Position 10 Position name T T XAR 1 BL Sequence DNA RNA Conc a IDofblank Posi in factor l il T 11 1 l1 Lt Assign a column from the data sheet Select the type of oligonucleotide that contains the sequences Press start test to load the first DropPlate96 on the DropSense96 and measurements will Start Page 49 5 1 5 Extra quantification of fluorescent labeled nucleic acids Measuring the labeling efficiency of fluorescent tagged probes before micro array hybridization eliminates potentially flawed samples and improves experiment effectiveness The full spectrum analysis with the DropSense96 allows measurement of nucleic acid absorption at 260 nm while
25. checks to ensure that it is functioning within specifications This quick test is identical to the quick systems check described in section 6 3 When a part of the system is out of specification then a warning screen will appear Please contact your distributor for a recalibration of the DropSense96 Warning Warning Pump0 has a decrease of pressure of 9 1 8 0 to 8 0 gt out of range Reference Pixel Position 2 324 out of range max 2 000 Reference Pixel Amplitude 0 001 Pump1 has a decrease of pressure of 3 1 in range max 1 000 8 0 to 8 0 gt in range Reference Pixel Dark 0 004 in range max 0 160 6 3 Potassium Dichromate test The DropQuant software includes a performance validation procedure as an easy check up of the performances of the DropSense96 The check is based on the measurement of an aqueous potassium dichromate K2Cr2O solution with a verified concentration commercially available from life science or spectroscopy companies It is recommended to use a highly concentrated potassium dichromate solution 0 4 0 8 OD at 350nm for a 1mm path length which is better suited for calibration of the short path length measurements performed in the DropSense96 It is good practice to check the instrument s performance every four months with a fresh vial of K2Cr2O When pressing the Potassium Dichromate test button a new screen pops up with Potassium dichromate automatically entered as sample
26. detecting the incorporated fluorescent dye at their absorption peak In a single measurement the DropQuant software calculates the nucleic acid concentration is expressed in ug ul and the dye concentrations in pmol ul NudeicAcids water based solvents incl TE PBS RT buffer zo lt gt Edit labels Cy3 Cy5 Alexa Fluor 488 SS Alexa Fluor 546 Enter a new dye Alexa Fluor 555 to the list Alexa Fluor 594 Alexa Fluor 647 Alexa Fluor 660 cy3 5 Cy5 5 DyLight 488 Dylight 549 ANCEL OPEN TRAY DyLight 594 DyLight 633 SS DyLight 649 Select dyes from DyLight 680 a pre defined list Continue on the Start new measurement screen Fill in this sheet as described in section 4 1 2 Choose as sample type Nucleic Acids and choose the option Labeled Other options like solvent can be selected as for other nucleic acid samples see 4 1 2 With selecting the option Labeled two drop down lists appear each containing a wide range of commercially available fluorescent dyes Use these drop down lists to select the appropriate dye or dyes If the nucleic acids have been labeled with only one dye choose None as the dye type for the second list To enter a new dye select the Edit labels button on the right side of the screen This will open a new screen for manual entry of the dye information Press the Add label button to enter the name and properties gi
27. duration to fill both measuring cuvettes on the DropPlates correctly This implies that the total reading time of an experiment is longer than a similar experiment with normal samples When the sample is still too viscous the dual measurement can fail This will be indicated by an orange or red color indication when the results are shown see further Heat the sample to 55 C and then gently vortex to minimize the viscosity and try to measure again Background correction Sample Sample Type Nucleic Acids Purified Proteins General UV vis lle Sample Material dsDNA Solvent water based solvents incl TE PBS RT buffer concentration factor nm 260 High viscosity concentration units ng ul Background correction default single point 1340 nm Background correction options A choice can be made between two types of background corrections The default background correction determines a linear fit through the 400 600nm part of the spectrum and subtracts this background curve from the full OD range The second option is to choose a single wavelength most common used wavelengths by Classic spectrophotometers are 320 340 and 405 nm The absorbance value of that wavelength will be subtracted from all wavelength data so the full spectrum will be set so that the background wavelength of choice will be zero During the data analysis the background correction can be altered again or t
28. factor specific to that sequence can be calculated and used in the concentration calculation With the DropSense96 it is posible to measure different oligo s in the same experiment using their specific sequence information The mode of action to quantify oligonucleotides is slightly different from that of other DNA RNA quantifications Continue on the Start new measurement screen Fill in this sheet as described in section 5 1 2 Choose as sample type Nucleic Acids and sample material Oligo An aditional selection between concentration calculation in ug ul or pmol ul be done on the right side of the Start new measurement screen This selection will also be used in the data analysis section Sample Sample Type Nucleic Acids Purified Proteins General UV vis Labeled Unlabeled Labeled Sample Material Oligo concentration factor Solvent water based solvents ind TE PBS RT buffer C High viscosity concentration units pn id P ual Selection of concentration calculation Other options like solvent labels and background correction can be selected as for other nucleic acid samples see 5 1 2 No information about the concentration factor on the right side is shown yet as this information will be calculated using the oligo sequence Press the next button to continue on the Plate layout screen When entering the blank and sample positions manually on the plate la
29. length 5 6 4 200 0 Path length Dual aam le volume pl i m 5 well plate SLE i BULL Corning 96 vell UV plate Greiner 96 ell UY Star Defaujt Set 4i Thermo Microtiter 96 vell plate as rename delete 96 vell NUNC plate Selection of brand and Create new type of 96well plate Path length selection When using DropPlate D disposables the user can select between a single or dual path length measurement see 3 1 2 The single measurement requires 1 ul of sample to perform a single pumping step and a small chamber measurement for high concentration sample analysis 2 110 OD 10mm equivalent absorbance The dual measurement requiring 3 ul of sample uses both measurement chambers for the analysis creating the large concentration measurement range 0 05 110 OD 10mm equivalent absorbance When using a DropPlate S only a single measurement is possible This consumable requires 2 ul of sample and performs a single measurement with a measurement range of 0 05 44 OD 10mm equivalent absorbance ave as a format When all the above selections have been made the user can save these in a new format for future use These formats are account dependent Only the lab manager account has the possibility to make a format that is available for all users The formats can be deleted renamed When all sample properties are entered press the next button on the tast bar below Page 86 5 3 3 DropPlat
30. of the column concentration 260 230 or 260 280 ratios can be chosen Furthermore the measurement description can be altered by pressing the edit button This overview can be exported to several formats like a CSV txt excel sheet or pdf file The data can also be send directly to a printer of choice Choice of data shown in the column Hesulls E Results IE al REPORT DISPLAY SINGLE WELL DISPLAY Concentration MEASUREMENT DATE TEST PERFORMED BY 12 06 2009 14 07 21 Guest Name Concentration INSTRUMENT ID PATH LENGTH blank 1 0 00 ng pl Double path length Bl sample 2 31 57 ng l EXPERIMENT NAME sample 3 62 51 ng yl 2 93 Experiment 2009 12 06 14h 07 Di sample 4 96 99 ng l 2 53 Ei sample 5 245 82 ng yl 2 48 Sam ple hee sample 6 600 74 2 68 data Gl sample 7 691 02 2 35 Hl sample 8 879 38 ng pl 2 38 7 2 0 230 A250 A320 A250 A230 Measurement info INFORMATION Full Sitwell measurement SOLVENT EACK TO DATABASE OPEN TRAY sample 20 Export and print options 711 85 ng yl EXPORT A2 sample 9 1914 18 ng pl 2 46 H20 sample 10 2552 65 ng pl 2 45 MATERIAL sample 11 3346 05 ng pl 231 dsDNA sample 12 451 49 ng yl 2 45 sample 13 721 18 ng pl 2 48 sample 14 859 99 ng yl 2 54 RAE sample 15 846 68 ng pil 235 sample 16 832 78 ng yl 2 32
31. the proper preferences in the sample definition box Page 63 Sample Select Purified proteins the button turn light gray upon selecting Labeled Make a choice between Unlabeled for general protein quantification or Labeled when a fluorescent label is present and the concentration needs to be determined Sample material Select the type of protein in this experiment Four sample types are available for purified protein analysis by selecting from the drop down list adjacent to Sample material For every selection the sample properties used for the concentration calculation are shown on the right side Sample ss antl on Acids __Generaluvi vis_ Sample properties labeled Sample Material Solvent i 3 51ngle protein EI 10 00 cl TE PBS RT buffer nm 280 concentration units mg ml Multiple protein Choice protein sample The default option General is a reference setting based on the assumption that the protein solution has an extinction coefficient of 1 so 1 OD 1 mg ml protein So a 1 10mg ml protein solution will have an A280 of 10 for a 10mm path length This option can be used if no extinction coefficient information exists for the protein sample and a rough estimate of protein concentration is needed Two protein references are included Bovine serum Albumin BSA and Human Immunoglobulin G IgG For BSA a prote
32. 2 0001 0005 005 0001 0 001 005 o o0 j DropPlatet H5 7 4097 0081 041 0437 0044 042 0 042 DropPlate1 A6 5 100 002 01 0108 0003 0 011 0 098 0 002 001 Page 94 5 3 5 4 Report display The report section gives an overview of the experiment information and all the measurement data in a table format The info on the data table can be altered by clicking on the header of the column Furthermore the measurement description can be altered by pressing the edit button This overview can be exported to several formats like a CSV txt excel sheet or pdf file The data can also be send directly to a printer of choice Choice of data shown in the column Results f Results OCreport GLOBAL gt lea RECALC DEBUG REPORT DISPLAY SINGLE WELL DISPLAY MEASUREMENT DATE TEST PERFORMED BY 2010 08 19 16 08 trinean Name A257 0 2mm OD y y A257 0 2mm OD Al Measurement PATH LENGTH blank 3 0 000 OD A257 1 0mm OD info DropPlate D Double path length B4 sample 26 0 192 OD A350 0 2mm OD EXPERIMENT NAME INSTRUMENT ID D4 sample 28 0 189 OD A350 1 0mm OD Experiment 2010 08 19 16 00 2010 061 F4 30 0 184 OD y DU DESCRIPTION H4 sample 32 0 184 OD 0 184 OD a 5 sample 33 0 125 OD 0 125 OD Sample D 2010 62 dilution serie Pot Dichrom i B5 sample 34 0 081 OD 0 081 OD d
33. 5 3 5 3 Singl welldispidy3iaa tud vie 92 5 3 5 4 REDON alee a ee ees 95 DIAGNOSTICS RD C 97 6 1 SYSTENUINEO AND SETTINGS nas can T 97 6 2 SEMPLE STING Ov 98 6 3 POTASSIUM DICHROMATE LES IM PE 98 6 4 QUICK SYSTEM CHECK 100 6 5 CHECK BARCODE READER OPTIONAL ACCESSORY ie potuto te eo i eoe i o e dt eoa ba oh 101 6 6 GENERATESTATUS ZIRI EE enisi nitate vus a ue tates mtf abe uia tones 102 6 7 CID CONTROLS onte teet 102 5 1 Overview 1 1 Product description The DropSense96 is a unique droplet plate reader combined with microfluidic DropPlate consumables This new generation of multichannel polychromatic spectrophotometer and consumables is suitable for high throughput UV VIS spectral analysis 220 750 nm of 1 3 microliter samples in life science labs with a manual or automated workflow The DropSense96 can also read standard 96well microtiter plates as alternative to the DropPlate consumables The system measures the absorption properties of samples and calculates the concentration levels of the substances e g nucleic acids or proteins Using the intuitive software the sample wells containing a specimen can be selected by a button click and the full UV VIS absorption spectrum of t
34. DETERMINATION eeeeeee nennen nennen nennen sterne stern tease rens 40 Du d Theory DNA RNA concentration and purity 40 4 5 1 1 1 DNA RNA concentration 40 SLL ODNARNA purty Geter 13575183539 LEM 40 5 1 2 Input or n cleic acid sample tat tet 42 5 1 3 DropPlate96 sample 46 5 1 4 Extra Quantification of oligonucleotides e e e E e st 47 5 1 5 Extra quantification of fluorescent labeled nucleic 0 50 5 1 6 StartmnedsUrelielt Pe E ET O E Oa 52 5 1 7 ante 53 5 1 7 1 displays asesino bea vc VS Vx ol ae ce en neve lca ad Fed d reru rdv oto rd et vds ves ot vM EUR 54 5 1 7 2 Colum displaye A 55 5 1 7 3 56 5 1 7 4 PRE DOLE CIS DAY ee 60 5 2 PROTEIN QUANTIFICATION E 62 2 2 4 Theory Protein concentration and purity determination esses 62 5 2 2 Starting a protein concentration
35. Dark 0 003 in range max 0 160 Pump Check Temperature is within the specifications 25 3 10 0 50 0 C Ambient light is within the specifications 3 009E 7A 0 000 0 1 000E 0A Periphery Pump has a decrease of pressure of 2 7 8 0 to 8 075 gt in range Pump has a decrease of pressure of 3 2 8 0 to 8 0 gt in range BACK CREATE REPORT After the check a pop up window shows the most important results An Excel report can be created using the create report button This report can be stored on a disk for future reference It is designed is a fast check so not all system parameters are checked The most important parameter that is not checked is the accuracy of the DropPlate positioning The Potassium Dichromate test is a better check of the positioning accuracy 6 5 Check barcode reader optional accessory The functionality of the internal barcode scanner can be tested using the check barcode reader button simply push this button and the barcode of the DropPlate in the instrument will be read and shown If there is no internal barcode scanner installed on the DropSense this option will not be active and displayed in grey 101 Use the Open Tray button to load a DropPlate with barcode in the system before starting the barcode check function The conversion time should be under 2 seconds If the scanner cannot read a code in 3 seconds then a time out warning is g
36. K 1918 5 10 INSTALLATION amc E 11 2 1 UNPACKING AND POSITIONING 11 2 2 INITIAL STARTUP 12 2 2 1 Computer requirements and software installation eee 12 2 2 2 Software IBS talla 12 2 32 3 Registering erem t oe dem eu nae O 13 2 2 4 Settng up the TOSEFUBTERE sio s tto o btt IN Mer 13 els uper 14 3 1 SAMPLE LOADING ON THE TRINEAN DROPPLATES Peta e E o emus 14 3 1 1 DropPlate General descripHIOn loi eese tino ett 14 3 1 2 Types of DropPlates tS amdiD sata booa tete tubae ito eb halal a nine aU 15 3 1 2 1 WHS DKO DP lat CHD seis M 15 3 1 2 2 Tie DEO ned cede ii 17 3 1 3 MOOG e goes Tolo 18 3 1 3 1 Loading DropPlates16 on the aluminum DropFrame cccccssssecccceeesecceceeeecceeeeesceeesaueeesseaeeessaaeeeeesages 18 3 1 3 2 Manual sample loading on a DropPlate ie tdi exea Cen ex EE are HR ee PER 19 3 1 3 3 Automated sample loading on EG Fee EE E E EYn oder va
37. K to continue Cancel Load the first DropPlate DropPlate1 on the tray and press The tray will be pulled in and the measurements will start Press Cancel if the measurement should not take place The software will return to the main menu The progress of a measurement can be observed in the small measurement screen showing wells that have been measured indicated by a green color and done wells being measured orange and indicating busy and wells that are still waiting to be measured white color and indicating To do Measurement gt Crinean ABORT TEST Page 52 5 1 7 Data analysis When the full measurement is done following screen appears bone boe Fone Bore fore E Please remove disposable from tray You can take the DropPlate96 of the microplate tray and press OK Now the software will generate the full spectral scan of all samples and calculate the concentration of the nucleic acid sample For safety reasons the microplate tray with the DropPlate96 will be pulled in automatically after 30 seconds and the measurements will be displayed Several display options can be chosen As default an 96well overview of the measurements is given Detailed analysis with setting options can be done in the single well display as explained in section 5 1 7 3 Type of display for analysis Post Proce
38. Nr Name Description sample 17 958 34 ng pl 254 1 HewDeopPiate sample 18 981 27 ng yl 2 45 sample 19 886 61 ng yl The Export button opens a new screen to create a data report as desired In the results export sheet a selection of columns can be made to be included in all three report types The order of the columns can be altered with the move up and move down buttons A preview of the report table is displayed at the bottom of the screen The export file type can be selected by clicking on the preferred file type Furthermore the information from the summary experiment description experiment name solvent and an overview of the plate layout can also be included by activating the include summary button The report design can be saved in a format for future use by clicking the save as button Page 60 Column selection Select elements to Export port Results Available columns Selected columns Source Plate ID DropPlate Position Gincilude Summary Export fil Source Position Sample name Ed include 96 well plate layout Export type DropPlate ID Pump selection Concentration ng ul A open new Excel file X Time A230 OD p Conc factor A260 OD Instrument ID A280 OD direct print Path length mode A260 A230 All Absorbance Values 10mm A260 A280 All Absorbance Values 0 5mm ME Material f Preview table Preview
39. abeled Other options like solvent can be selected as for other proteins see 4 2 2 Sample TT Sample Material BBA 6 67 nm 1280 Enter a new dye High viscosity concentration units mg ml to the list s a 1 Cy5 Alexa Fluor 488 Set as default Alexa Fluor 536 DropPlate Type DropPlate type AlexaFluorsoa Alaxa Fluor 647 Cy3 5 Cy5 5 Solvent water based solvents incl TE PBS RT buffer yLight 488 CANCEL yLight 549 594 Light 633 Dytight 643 Select dyes from L L DyLight 680 a pre defined list With selecting the option Labeled two drop down lists appear each containing a wide range of commercially available fluorescent dyes Use these drop down lists to select the appropriate dye or dyes If the protein has been labeled with only one dye choose None as the dye type for the second list To enter a new dye select the Edit labels button on the right side of the screen This will open a new screen for manual entry of the dye information Press the Add label button to enter the name and properties given by the manufacturer like the appropriate correction factors can be entered The 280 nm correction will be utilized for protein concentration calculation when the respective dye is selected To remove a dye select the dye and press the Remove label button Pre defined dyes
40. al information When selecting a DropFrame96 in the table colored blue the 96well display underneath will show an overview of this particular DropPlate96 Page 30 Layout of selected DropFrame Plate Layout 2 Plate Layout DropPlate DropPlate 2 Fi Select click mode Select sample type in well CANCEL OPEN TRAY se RR lt PREVIOUS START TEST gt PLATE LAYOUT PLATE LAYOUT Enter source plate information Export overview of plate in xls txt or csv Select click mode Select click mode contains two choices insert or view update The insert mode will directly insert the information below type well well name in the selected well in the 96 matrix on the left So the user can change the information for a well first and by clicking on the well the information is stored With the view update mode the user can click on a well and obtain the information of this well without changing it If the information of the well needs to be chanced the user can change the info type well well name and then click update above right on the screen Type well Three well types can be chosen by clicking on the colored square Empty grey Blank blue Sample red The yellow reference cannot be chosen yet but will be used in future applications The empty option can be used to erase errors However a safety procedure is implemented in the software to avoid erasing blank wells
41. al shape and concentration calculation is shown without blank compensation In this mode the shape of the blank spectrum itself can be analyzed When the blank subtraction is turned off all other screens and the report section will generate data without blank subtraction e Background correction the background correction method can be selected in the start new measurement sheet section 5 1 2 The background information is derived from the RAW OD spectrum measured In the default setting a linear fit through the 400 600 nm region of the transmission spectrum is subtracted from the full spectrum while in the single point mode the background level of a wavelength of choice is subtracted from the full spectrum The manual selection on the single well display offers the option to chose between the default method a single wavelength of choice or no background correction This allows to verify if the background is not near zero as indicator for turbidity or presence of debris Note that changes in the background correction will be used in the other screens and report section In the report section the RAW OD value of the wavelength shown in the single point background correction for instance RAW A405 10 mm can be included in the report table to export The single well display offers a quick export to XLS function located underneath the spectral view This function is limited to an export to Excel showing all the spec
42. an intermediate path length of 0 5 mm which is suitable for the high speed analysis of samples with a more limited concentration range 0 05 44 OD 10 mm equivalent absorbance Alternatively a standard 96 well microtiter plate can be used instead of the DropPlate consumables A pulsed xenon flash lamp provides the light source and a multichannel spectrometer with 4 linear CCD arrays in a row is used for high speed analysis of the light that passes through 4 samples simultaneous The DropSense96 is connected to a PC for controller software data display and additional archiving or data printing Page 1 4 Theory of optical system A functional diagram of a DropSense96 spectrophotometer module is presented here t lamp mirror ting gren detector The DropSense96 optical core includes the following A pulsed xenon flash lamp is used in the DropSense 96 to provide a wavelength range from 200 to 1000 nm The sample holder which contains the sample allows the light to pass through and does not affect the measurement A variety of materials are used depending on the wavelength to be monitored The DropPlate is commonly used as sample holders The white light goes through the sample and falls upon a cylindrical lens Light is transmitted through the lens and falls upon a grating structure that reflects the different wavelength bands to the upper part of the lens Part of the lens surface has
43. aned underneath stay fixed within the other data analysis screens and the export options Page 78 e Change scale Both the scales of the X axis and the Y axis can be altered for a detailed view by changing the highlighted wavelength at the beginning and end of the scale The setting autoscale has to be turned off when changing Y axis Note that the report option All absorbance values 10mm will show all the absorbance values within the selected wavelength range of the X axis So manual changing of the X axis scale will change the info in the report e Blank subtraction When using a blank reference sample it is automatically subtracted from the sample absorbance before concentration calculation This feature can be turned off so the spectral shape and concentration calculation is shown without blank compensation In this mode the shape of the blank spectrum itself can be analyzed When the blank subtraction is turned off all other screens and the report section will generate data without blank subtraction e Background correction the background correction method can be selected in the start new measurement sheet section 5 1 2 The background information is derived from the RAW OD spectrum measured In the default setting a linear fit through the 400 600 nm region of the transmission spectrum is subtracted from the full spectrum while in the single point mode the background level of a wavelength of choice is subtrac
44. ar color legends are used as with the DropPlate D A black background indicates a good measurement while a red color inducates a failed measurement As the DropPlate S only contains one micro cuvette no orange color legend is used 5 2 7 2 Column display Choice of spectra Choice of data shown Spectrum name and data shown on display under spectrum of sample 4 in well D1 Results re Results REPORT DISPLAY Spectrum OD 10g J Concentration LJ au 1 1 1 230 250 275 300 325 350 375 400 230 250 275 300 325 350 375 400 7 Cis af rna Oe ue lt 4 m 2 lt P Column asses ee alg bee tration T 01 ea selection centration 1 21 230 250 275 300 325 350 375 250 275 300 325 350 375 400 230 250 275 300 325 350 375 400 Hl sample 8 LA JE T T2 1 O El us mE mra v BACK TO DATABASE alle Page 76 Gives an overview of all measurements per column Clicking on another column opens all spectra of this column Underneath every spectrum the sample name and data are displayed A data selection can be done using the scroll button above the spectra A choice can be made between concentration or A260 A280 ratio The view of the spectra can be changed from OD 10mm to the combined view OD long OD short using the Spectrum Type button 5 2 7 3
45. ased solvents incl 85 RT buffer water glycerol 5 10 water DMSO 5 10 Choice solvent Extra button for high viscous samples Page 65 solvents containing low amounts of salts and buffers are grouped in one option since these substances don t affect the pumping properties An extra button for highly viscous samples can be used so the pumps will work at higher pressure and longer duration to the measuring cuvettes on the DropPlates correctly Viscous protein samples should be handled with care as high viscosity can hamper both precise pipetting of the samples and transport of the sample in the microfluidic DropPlate16 When the sample is still too viscous the measurement can fail This will be indicated by an orange or red color indication when the results are shown see further Furthermore proteins can denaturate precipitate or aggregate leading to non homogeneous samples Background correction A choice can be made between two types of background corrections The default background correction determines a linear fit through the 400 600nm part of the spectrum and subtracts this background curve from the full OD range The second option is to choose a single wavelength most common used wavelengths by classic spectrophotometers are 320 340 and 405 nm The absorbance value of that wavelength will be subtracted from all wavelength data so the full spectrum will be set so that the backgro
46. at are still waiting to be measured white color and indicating To do Measurement Cf Inear ABORT TEST Page 73 5 2 Data analysis When the full measurement is done following screen appears crinean lel la alla about TEST mm ss E E Please remove disposable from tray You can take the DropPlate96 of the microplate tray and press OK Now the software will generate the full spectral graph of all samples and calculate the concentration of the protein samples For safety reasons the microplate tray with the DropPlate96 will be pulled in automatically after 30 seconds and the measurements will be displayed Several display options can be chosen As default an 96well overview of the measurements is given Type of display for analysis Results J OD Long OD Short Choice of spectra shown on display BACK TO DATABASE OPEN TRAY Page 74 5 2 7 1 96well display Gives an overview of all spectra and the concentration of the nucleic acid calculated from A260 See example above In the standard mode all spectra are shown normalized to a 10mm path For a DropPlate D measurement a combined spectral view of both path lengths can be shown by selecting OD long OD short in the Spectral Type button Detailed analysis of one sample can be achieved by clicking on the sample spectrum opening a detailed figure of this spectru
47. ata c5 sample 35 0 124 OD 0 124 OD SOLVENT DS sample 36 0 080 OD 0 080 OD water based solvents incl TE PBS RT buffer 5 sample 37 0 121 OD 0 121 OD MATERIAL F5 sample 38 0 080 OD 0 080 OD General UV vis Pot Dichrom 65 sample 39 0 120 00 0 120 OD HS sample 40 0 081 OD 0 081 0D A6 sample 41 0 055 OD 0 055 OD B6 sample 42 0 036 OD 0 036 OD VIS Ed C6 sample 43 0 056 OD 0 056 OD D6 sample 44 0 037 OD 0 037 OD 1 DropPlate 1 E6 sample 45 0 053 OD 0 053 0D sample 46 0 035 OD 0 035 OD BACH TO DATABASE TRAY sample 47 0 054 OD EXPORT 0 054 OD Export and print options The Export button opens a new screen to create a data report as desired In the results export sheet a selection of columns can be made to be included in all three report types The order of the columns can be altered with the move up and move down buttons A preview of the report table is displayed at the bottom of the screen The export file type can be selected by clicking on the preferred file type Furthermore the information from the summary experiment description experiment name solvent and an overview of the plate layout can also be included by activating the include summary button The report design can be saved in a format for future use by clicking the save as button Page 95 Column selection Ww Select Select elements to Export Results Available columns Selec
48. ave as button Page 81 Column selection Select select elements to Export Results Available columns Selected columns Export Source Plate ID DropPlate 10 indude Summa SING LM Scones Basi TY Export file type ource Postion ropPlate Position Include 96 well plate layout selecti on Pump Sample name d d Time Concentration mg ml openinexistingExcelfile Emm A260 A280 pat Instrument ID c Maj Material Path length mode B8 9 direct print All Absorbance Values 10mm Preview table Preview DropPiate Sample Concentration AZ amp D A28D Material Pasition impmi Drepframeil A1 blank 1 ig Drapframel 81 sample 2 237 os Drepframel a sample 3 3 03 os a Create report DrapFrame 1 D1 __4 233 0 54 De Dropframel 1 sample 5 3 03 dw DrepFramei F1 299 tga a 1 d U Glinclude outliers EXPORT RESULTS Save as format Page 82 5 3 General UV Vis spectrophotometry 5 3 1 Introduction In the general UV Vis application the DropSense96 functions as a conventional spectrophotometer An absorbance scan from 230 to 750 nm of any liquid samples can be analyzed enabling simple identification of absorption peak heights and positions An unlimited number of wavelenghts can be designated in advance for absorbance monitoring and inclusion in the report
49. be changed using the Spectrum Type button Single well display Gives a detailed view of a sample spectrum and all additional sample data next to the spectrum A sample can be viewed by clicking on the name of the well or sample in the select input well table or by clicking on a well in the 96well overview Results Results REPORT DISPLAY Select Input Well blank 1 Sample i sample 10 sample sample sample _ sample sample sampl 11 12 13 14 L5 16 sample 17 sample 18 sample 19 sample 20 sample 21 m mmm mumumumumm mummumummum mamumuumum mammummumum SOc BACH TO DATABASE a D Choice of spectra shown on display SINGLE WELL DISPLAY OD 1 0mm OD 0 2mm autoscale 300 350 400 450 500 550 600 650 700 Ea subtract Wavelength QUICK EXPORT MLS EXPORT DATA Change grafic X and Y scale EXPORT Show hide cursor with display of wavelenght and OD GLOBAL DEBUG Oc report RECALC wavelength nm 257 1 0mm Absorbance OD 0 2mm Absorbance 0D A257 0 2mm OD Al 0 000 OD 1 Sample information B2 0 12400 818500 Change settings blank background correction default iingie point aos CHANGE PARAMETERS Export of spectra and sample data to excel Extra tools are included for detailed
50. been coated with a mirror and reflects the wavelength bands back upon a highly sensitive CCD camera chip Data are transmitted to the PC where data analysis software verifies and calculates the diminution in values for certain wavelengths This enables to plot a full scan a micro seconds of time Page 8 1 5 Applications The DropSense96 couples ease of use with high speed UV VIS spectrophotometry The small sample requirement using the DropPlates makes the DropSense96 ideally suited for measuring e Concentration A260 nm and purity of nucleic acid samples DNA RNA oligo s e Fluorescent dye labeling density of nucleic acid microarray samples e Purified protein analysis A280 nm e Quantification of fluorescent dye labeled proteins e API quantification Active Pharmaceutical Ingredient e General UV Vis spectrophotometry 1 6 Components 1 6 1 Front panel The front panel contains the central positioned microplate drawer and three indicator lights Back panel Front panel Indicator lights DropPlates on a 96 format frame Power Computer port Microplate drawer racamae Indicator lights Three indicator lights are present Green Orange and Red light For DropSense96 instruments following light color indications are used 1 Green orange and red light combined during DropSense96 start up all three lights will flash simultaneously for about 10 seconds 2 Green and red light DropSense96 i
51. ctor 50 00 nm 260 High viscosity concentration units Background correction default single point 1340 nm Background correction options A choice can be made between two types of background corrections The default background correction determines a linear fit through the 400 600nm part of the spectrum and subtracts this background curve from the full OD range The second option is to choose a single wavelength most common used wavelengths by Classic spectrophotometers are 320 340 and 405 nm The absorbance value of that wavelength will be subtracted from all wavelength data so the full spectrum will be set so that the background wavelength of choice will be zero During the data analysis the background correction can be altered again or turned off for detailed analysis Page 85 DropPlate selection Depending on the application or sample concentration range a choice of DropPlate D or DropPlate S can be made Both DropPlates are explained in detail in section 3 1 Alternatively a standard 96well microtiter plate can be used This selection requires additional information like path length or sample volume combined with plate brand and type in order to calculate the correct path length Additional plate brands and types can be entered by selecting lt Create new gt from the drop down list Selection of path length or volume DropPlate Type DropPlate type Standard 96 well plate path
52. d on the DropQuant CD included in the package upon arrival New accounts at the user level can be made and passwords can be given A new user account has only limited access to the measurement database and can only view his own measurements in the database However when user needs to be allowed to see all data in the database then the Access to all experiments should be turned on Other accounts in grey are not accessible The Guest account is the default log in setting and can be used without a password This account has very limited access to the DropQuant software functions Accounts Accounts trinean Name 1 T eryrce lab manager Level User JAccess to all experiments Password Edit Measurement Database location Alter the location of the raw data measurement database by using the explorer function here o Edit formats In the user software settings can be saved for future use by saving them as a defined format like import and export formats In this section an overview of all used formats will be displayed and a selection of formats can be made by the lab manager so all other user accounts can see and use these formats Export settings Export all formats user database These will be saved in a separate folder This folder can then be used on another PC with installed DropQuant software Page 26 using the import settings button All saved formats will be activated on the
53. d reproducibility Meander shaped reservoir Input well Alignment hole Extra safety reservoir vaccuum pressure O Page 14 Two DropPlate formats can be read on the DropSense96 spectrophotometer e DropPlate16 this small version of the DropPlate consumable has 16 input wells for a low to medium throughput mode For low sample numbers 1 to 6 DropPlates can be assembled on a aluminum DropFrame creating a flexible set up measuring 1 to 96 samples simultaneously For higher sample numbers assembly of 6 DropPlates on a frame will generate a 96well microfluidic plate with identical outer dimensions as a standard 96well plate assuring compatibility with liquid handling robots e DropPlate96 the 96well Microtiter plate sized DropPlate96 is suited for high throughput use This consumable is ideal for automated workflows as it is easy stackable and bar coded for sample tracking The DropPlate96 is compatible with liquid handling robots SBS standards and permits easy loading and preservation of samples and measurement of the optical absorption of the droplet Note It is not necessary to fill all 16 input wells to perform a measurement A single measurement can be done and unused positions on a DropPlates can be filled in another measurement until all input reservoirs have been filled 3 1 2 Types of DropPlates S and D Two DropPlate versions have been developed with slightly different microfluidic structures
54. d using a small path with a large path length for length for high concentrated low concentration samples samples Depending on the required OD range a choice can be made for a single or dual chamber measurement The single chamber measurement has a path length of 0 2mm with an OD range of 1 75 OD for a 10mm equivalent path This single chamber mode is ideally suited for highly concentrated samples The dual chamber measurement option contains a dual path length 0 2 and 1mm measurement creating an OD range of 0 05 110 OD for a 10mm equivalent path The dual path length option has a wider measurement range then the single chamber mode and is suited for the majority of biomolecule samples A selection for the single or dual chamber measurement can be done in the DropQuant software Sample size requirements Although the dispensed sample volume is not critical it is essential that a minimal amount of sample is dispensed for correct filling of the measurement chambers allowing precise measurements Extensive testing indicates that sample volumes of 1 5 ul for a single chamber measurement and 3 ul for a dual chamber measurement are sufficient to ensure reproducibility Although this volume range takes a pipetting error into account it is best to use a precision pipettor 0 5 5 ul with precision tips to ensure that sufficient sample is used Page 16 3 1 2 2 The DropPlate S The DropPlate S looks very similar to the DropPlate16 D howeve
55. d using the A260 A280 ratio A A260 A280 ratio lt 1 indicates pure protein whereas a higher value indicates nucleic acid contamination An alternative method for protein quantification is a colorimetric protein assay such as a BCA Bradford and Lowry assay These are commonly used for quantification of protein solutions and cell lysates These types of assays can easily be performed with the DropSense96 using the general UV vis mode in the software Quick export of the data to excel allows fast generation of a standard curve and concentration determination 5 2 2 Starting a protein concentration measurement Pressing the new measurement button on the main menu opens following screen New measurement X 20911 _ Start new measurement Crinean Experiment definition Sample definition Sapte ype ele Experiment Experiment name Experiment 2010 10 20 15 52 Sample Sample Material dsDNA zi Solvent water based solvents incl TE PBS RT buffer concentration iat concentration units ngu Select DropPlate type Background correction default and path length single point 1 05 nm DropPlate Type DropPlate type DropPlate D 1 Path length Save as Format Task bars Default save save as rename delete OPEN TRAY NEXT gt All options on this screen are described in detail in section 4 1 2 For measurement of proteins select
56. e the 1mm pathlenght is used up to OD 1 OD 10 when normalized 1 cm path Above 1 OD at 1 mm the concentration is calculated with the 0 2 mm path absorbance measurement During the measurement cycle the transport of the sample through the microfluidic structure is monitored by continuous spectral measuring This way errors occuring during the process can be detected and displayed on the screen This allows a thourough and robust analysis of all measurements A color legend is used as illustrated on the previous page When using DropPlate S consumables the software will normalize the single cuvette measurement 0 5mm path to a standard 10 0 mm path Similar color legends are used as with the DropPlate D A black background indicates a good measurement while a red color inducates a failed measurement As the DropPlate S only contains one micro cuvette no orange color legend is used 5 1 7 2 Column display Choice of spectra Choice of data shown Spectrum name and data shown on display under spectrum of sample_4 in well D1 Post Processing Results x GLOBAL DEBUG ri REPORT DISPLAY A WELL DISPLAY COLUMN DISPLAY 96 wt DISPLAY Concentration 4200 4230 4260 4280 400 230250 275 300 325 350 375 400 Column selection 1 2 3 4 3 6 7 8 9 sempe 5 Boemie BACK TO t CHANGE pet OPEN TRAY EXPORT DATA 55 Selected well f
57. e96 sample layout Pressing the next button on the Start new measurement screen will open the next screen where information on sample data and DropPlates96 layout can be entered The positioning information of samples or blank can be entered manually or can be imported from an excel txt or CSV file Both import methods are described in detail in section 4 1 4 Number of DropPlates96 Activate barcode reading Plate Layout nean Plate Layout Selected DropFrame Manual well definition overview gt 2 EJEIEIEELEJERETETETETES i SIBEIEIEL EIE EL EL ELE DEBEBEHBBEDBEUD EJEIBLEELE EL EIL EL EI EJ EI EIEIBIEID E EEELELEL EI o e Overview well names import export functions Page 87 5 3 4 Start measurement Press the Start Test button on the task bar of the Plate layout screen This will open the microplate tray of the connected DropSense96 and the following screen will appear Measurement 1 2 3 4 5 6 7 8 E 10 1i 12 e crinean i iii ABORT TEST Please insert disposable nr 1 DropPlate 1 in the tray and press OK to continue Cancel Load the first DropPlate DropPlate1 on the tray and press OK The tray will be pulled in the measurements will start Press Cancel if the measurement should
58. easurement is possible This consumable requires 2 ul of sample and performs a single measurement with a measurement range of 10 1200 ng ul dsDNA Page 45 Save as a format When all the above selections have been made the user can save these in a new format for future use These formats are account dependent Only the lab manager account has the possibility to make a format that is available for all users The formats can be deleted renamed When all sample properties are entered press the next button on the task bar below 5 1 3 DropPlate96 sample layout Pressing the next button on the Start new measurement screen will open the next screen where information on sample data and DropPlates96 layout can be entered The positioning information of samples or blank can be entered manually or can be imported from an excel text or csv file Both import methods are described in detail in section 4 1 4 Number of DropPlates96 v Plate La yout mean Plate Layout a Add DropPlate DropPlate 1 SEE Pe DropPlate 2 s 4 Manual well definition EXPORT IMPORT NE PREVIOUS STARAT TEST gt PLATE LAYOUT PLATE LAYOUT Overview well names Import export functions Selected DropFrame overview Page 46 5 1 4 Extra Quantification of oligonucleotides oince oligo s have defined sequences their molecular weight molar extinction coefficient and the concentration
59. ection will be used in the other screens and report section In the report section the RAW OD value of the wavelength shown in the single point background correction for instance RAW A405 10 mm can be included in the report table to export The single well display offers a quick export to XLS function located underneath the spectral view This function is limited to an export to Excel showing all the spectra selected on the single well display and a fixed selection of columns with all necessary data from the selected samples e Lf Inear DropSense REPORT 0 85 gp 1 0mm amp 0 2mm Absorbance OD 10 1 56 6 SEELA eT LLLLETLEALTTI SE Sr Neer ber NC ano Wavelength 638 A638 A638 411 411 411 Raw A405 A405 Raw A405 DropPlate ID Sample name 5 ion Br 10mm mo 0 2mm EA 1mm mes 10mm E M 0 2mm OD 1mm 10 000000 0 200000 E 000000 DropPlate 1 1 E5 044 14 0004 004 Q014 014 0034 034 000 002 0 003 0 048 0 003 0005 005 ee ee ee ee DropPlatet 65 1 005
60. efault of blanks Ar of rows to skip header not included q Singa blan TI header Multiple blanks fave as Source Plate DropPiate io gare DropPlate 10 of blank Source Postion P mw Position EL I Blank Position t Sample name a OR name of blank used in file press enter to confirm gt Single blank converted sample s 0 more info identification selecdied sample s 0 select all deselect all Source DropPlate DropPlate j DropPlate Blank Position iB Position ID of blank Position CANCEL Page 39 5 Applications 5 1 Nucleic acid quantification and purity determination 5 1 1 Theory DNA RNA concentration and purity determination 5 1 5 1 1 1 DNA RNA concentration measurement Nucleic acid samples can simply be checked for concentration and quality using the DropSense96 spectrophotometer Nucleic acids have an absorption maximum at 260 nm A260 and the spectrographic reading at this wavelength is the most common method for detecting dsDNA ssDNA RNA and oligonucleotides in a solution The nucleic acid concentration is calculated using the Beer Lambert law see below which predicts a linear change in absorbance with concentration Using this equation with a 1 cm path length an A260 reading of 1 0 is equivalent to 50 ng ul dsDNA 33 or 40 ng ul single stranded RNA Beer Lambert Law A c Where A
61. elected wavelength and its OD value 10 mm is displayed 77 e Path length selection Above the spectral view a choice can be made between OD 10mm or OD 1 0 mm OD 0 2mm for a DropPlate D measurement or OD 0 5mm for a DropPlate S measurement Note that the Y axis scale will automatically adjust to the proper OD values When selecting OD 1 0 mm OD 0 2mm the 1 0mm OD spectra will be shown with a full line while the 0 2mm OD spectra will be displayed using a dotted line e Overlay spectra when pressing the CTRL button on the computer keyboard select two or more samples from the select input well table to overlay the selected spectra When using the shift button and selecting two samples all samples between the selected will also be displayed on the screen crinean Results REPORT DISPLAY SINGLE WELL DISPLAY COLUMN DISPLAY 96 WELL DISPLAY Select InputWell hide cursor wavelength 10 10mm Absorbance OD 345 blank 1 autoscale Concentration mg ml sample_ 8 sample 9 sample_ 10 sample 11 sample 12 sample_ 13 10mm Absorbance OD sample 14 300 320 340 360 subtract Wavelength st background correction QUICK EXPORT TO XLS default single point 405 nm BACK TO EXPORT DATABASE Additional tools are included to change the analysis settings of the measurements Changes made within these tools expli
62. emplate button to open an example sheet with defined columns These columns can be deleted or others can be added to create a customized import excel sheet Import format design After entering excel txt or CSV data a import format has to be designed The default general format has been designed in combination with the excel template This excel template can be viewed by clicking in the open xls template The small button next to general opens the detailed formatting options Now several drop down buttons are shown devided in three sub groups source description DropPlate description and Blank information Next to the latter sub group several buttons are foreseen to add new formats save delete or rename them Using the add button a new format can be defined compliant with the user specific imported data file import Samples Import Samples Open format open xls template 2 details import format Default 5 Nrofrowsto o header not incuded header 27 Source Plate ID P cumi DrapFrame iD FP wm Y Source Postion uM Position mM LI 5ample name cls converted sampled more info selected sampleg select all deselect all Source Source Dropframe Drop sme Sample DropFrame Blank PlateiD Position 10 Pos jon tree name ID of blank Position 1 T 1 7 0 E Source description DropPlate description Blank descr
63. enerated Genomic DNA lambda DNA and viscous solutions of highly concentrated nucleic acids are common examples that require careful attention to ensure homogeneity before sampling Furthermore proteins can be subject to denaturation precipitation and aggregation and therefore may require special handling to ensure sample homogeneity Sample evaporation is minimized due to the meander shaped sample reservoir that takes up the sample automatically during sample dispensing This design avoids large measurement inaccuracy due to evaporation effects and sample concentrations remain constant for about 2 hours Page 20 3 2 DropSense96 Mode of action 3 2 1 Instrument start up The power switch is located on the back panel of the DropSense96 Push the rocker switch to the ON position The instrument automatically pulls in the microplate tray when the tray was in the open position When the DropSense96 instrument is powered on double click the DropQuant software icon on the computer desktop to start the program Once logged in a first message will indicate that the USB connection is being made the instrumental hardware settings are being read and diagnostic checks are performed to ensure correct functioning Checking Optics Checking Pumps Updating values If the pumps spectrophotometer don t perform as expected during the diagnostic check up an information window will automatically pop up see examples belo
64. ft in wavelength accuracy 1 nm can result in 0 2 change in the 260 280 ratio The solution properties pH and ionic strength can also affect the 260 230 and 260 280 ratio s Therefore we recommend using a buffered solution like TE pH 8 0 as both the nucleic acid diluent and the blanking solution during the measurement Pure water often has an acidic pH lowering the 260 280 ratio while TE buffer has an intrinsic UV absorption below 240 nm Page 41 5 1 2 Input of nucleic acid sample data Continue on the Start new measurement screen emeni Start new measurement Experiment definition _ Sample definition Select DropPlate type and path length Save as Format 25 Task bars NEXT CANCEL Experiment definition The default experiment name includes date and time for easy chronologic overview of the database The experiment name can be changed and a experiment description can be added Both are displayed in the database section Sample Type Select Nucleic Acids on the Start new measurement Menu the button turns light gray upon selecting Labeled Make a choice between Unlabeled for general nucleic acid quantification or Labeled when a fluorescent label is present and the labeling efficiency needs to be determined Sample material Select the type of nucleic acid in this experiment The user can select dsDNA ssDNA RNA and Oligo
65. ge 17 account it is best to use a precision pipettor 0 5 5 ul with precision tips to ensure that sufficient sample is used Overview of DropPlate types DropPlate 16 DropPlate96 DropPlate S DropPlate D 16 input wells aluminum DropFrame 2 ul samples OD range 0 1 44 OD dsDNA 5 2200 IgG 0 15 33 mg ml 16 input wells aluminum DropFrame 3 ul samples OD range 0 1 110 OD dsDNA 5 5500 ng ul IgG 0 15 95 mg ml 96 input wells Full disposable 2 ul samples OD range 0 1 44 OD dsDNA 5 2200 ng ul 0 15 33 mg ml 96 input wells Full disposable 3 ul samples OD range 0 1 110 OD dsDNA 5 5500 ng ul IgG 0 15 95 mg ml 3 1 3 Mode of action 3 1 3 1 Loading DropPlates16 on the aluminum DropFrame Up to 6 DropPlates16 can be placed on a DropPlate frame creating a full 96well plate with standard 96well plate dimensions SBS standard The DropPlates16 can only be placed in one way on the DropFrame due to the alignment pins and orientation triangles on the frame It is recommended to put the DropPlates16 on the frame before loading the liquid samples The DropFrame has been made black to aid the visual inspection of loaded or empty input wells and associated sample reservoirs This specific design of the DropFrame minimizes human errors during sample loading Stackable DropPlate 96 frames Alignment pins for DropPlate16 positioning Orientation triangle on
66. he samples is displayed and stored on the connected PC The Trinean DropSense96 platform can be fully integrated into a automated set up by combining it with standard liquid handling robots 6 1 2 Intended use of the Instrument The TRINEAN DropSense96 droplet spectrophotometer is intended for research and clinical use by trained personnel The instrument is intended for the measurement of absorbance of samples in DropPlates consumables and standard 96well microtiter plates 1 3 Overview technology Using the combination of the Trinean DropSense96 with DropPlate consumables samples are directly loaded onto the DropPlates containing hydrophilic input wells and channels for immediate sample intake and evaporation free storage The following microfluidic DropPlate consumables can be used the small DropPlate16 has 16 input wells for a low to medium throughput mode and the 96well microtiter plate sized DropPlate96 is for high throughput use Both are compatible with liquid handling robots SBS standards and permit easy loading and preservation of samples and measurement of the optical absorption of the droplet with variable path lengths Two microfluidic alternatives are available the DropPlate D Dual has two superposed microcuvettes to perform a dual path length measurement 0 2 and 1 0 mm covering a large OD measurement range 0 05 110 OD 10 mm equivalent absorbance The DropPlate S Single contains a single microcuvette with
67. hods are described in detail in section 4 1 4 Selected DropFrame overview 9 2 4 Number of DropPlates96 Activate barcode reading x Plate Layout cinean Plate Layout 4 Add DropFrame Read bar code RemoveDropFrame Manual well definition select dick mode E insert Y l Position Bp Type well Blank P d Well name blank Source Plate ID Source Position 5 6 a 10 12 gt EXPORT IMPORT CANCEL OPEN TRAY SAMPLES SAMPLES lt PREVIOUS START TEST gt Overview well names import export functions Extra Measuring multiple proteins with different extinction coefficients Using the DropSense96 and its DropQuant software a collection of different proteins with different UV absorbance properties can be measured using a single experiment definition Continue on the Start new measurement screen Fill in this screen as described in section 4 7 2 Choose as Sample type Purified proteins and as Sample material Multiple proteins Other options like solvent can be selected as for other proteins see 4 2 2 No information about extinction coefficients is displayed on the right side as this information is protein dependent Entering the extinction coefficient of the protein collection can be done manually or be imported Page 68 J General BSA IgG Single protein Multiple proteins
68. imited access to the software DropQuant erinean DroepQuant for DropSense 96 Help button SYSTEM INFO OPEN TRAY MEASUREMENT NEW AND SETTINGS xtd t hen DATABASE MEASUREMENT gt 1 it Log in information M EC switch log in button Go to the measurement menu 1 X 1 Connect to the 96 Open Close tray Open measurement L database Extra options for lab manager log in 4 1 2 2 Main menu features Log off Use this button to change the account or to close the DropQuant software Connect This button is only shown when the software is not connected to a DropSense96 When connection is needed plug the DropSense USB cable in and press connect Open Close tray With this button the plate carrier can be opened or closed at any moment Helo Button This button opens the user manual in pdf form Page 24 System info and settings Using the login lab manager additional options appear The system info and settings button at the left bottom side of the main menu leads to a screen displaying several editing options and an overview of the systems information 4 1 2 3 System info and settings The Systems info and Settings button only appears when using the lab manager account or an account with similar entry level This button opens a new window containing two tabs for systems info diagnostic tools and software setting options System in
69. in sample concentration is calculated using the mass extinction coefficient of 6 67 at 280 nm for a 1 10 mg ml BSA solution For IgG a protein sample concentration is calculated using the mass extinction coefficient of 13 7 at 280 nm for a 1 10 mg ml IgG solution The option Other lets the user enter the extinction values of a single protein Two options can be selected e Enter the molar extinction coefficient M cm 1 and molecular weight MW in Daltons Da of the protein solution The appropriate e and the MW Da should be entered Page 64 and a software calculation of the extinction coefficient for 10mg ml solution E1 is performed and used to calculate the protein concentration e Enter the mass extinction coefficient L g 1 cm 1 for a 10mg ml 1 protein solution The appropriate extinction coefficient E196 should be entered before the measurement Finally when measuring a DropPlate96 containing several proteins with different extinction coefficients the multiple proteins option has to be selected For each protein the appropriate extinction coefficient E196 can be entered for precise concentration determination see 5 2 4 Solvent Select the solvent used for the protein samples to be measured This solvent information is used by the DropSense96 to fine tune the pressures used by the vacuum pumps guaranteeing precise and controlled filling of the microfluidic structures All water based water b
70. indows XP Service Pack 3 5 1 Potassium dichromate test Drop Quant ersion 0 4 154 AEA Quick system check P FCrentor version 1 0 1 Salite installed 127 Check bar code reader Drop uant Remote version lenat installed Disk cA Status Total diskspace MB 250056 Generate Status Zip File Free diskspace MB 149774 Dropsense DrapSense Serial Number 12010 0033 Mr Mesi 151 SYSTEM INFO Mica AND SETTINGS Nr Pulses Lamp 313066 Last Calibration 5 17 2010 Nr Pumpcycles pump 347 Firmware version 1 34 Hr Pumpcycles 1 334 Pump Time s pump 2195 Pump Time s pumpi 2054 OPEN TRAT This button will open up an extra screen containing two tabs The Edit Settings tab is explained in detail in section 4 1 2 2 and can be used by the lab manager to create new accounts change the location of the measurement database import or export created formats and settings The System Info tab contains a detailed description of the PC configuration like free disc space and DropQuant version installed detailed info on the attached DropSense96 serial number last calibration date lamp and pump info diagnostic checks and a ZIP function to save a general status of the Dropsense96 to be sent to the dealer for a first system analysis Page 97 6 2 Self testing Upon instrument start up or account log in the DropSense96 automatically performs diagnostic
71. iption Page 37 After giving the new format a name the columns as shown in the preformed table above must be assigned to the appropriate definition Source plate ID Source position sample name If the automatic imported table contains rows before the required data and or a table header then this information can be specified for an optimal table view The appropriate rows need to be selected containing source information and DropPlate information Import Samples ae example Delimiter Pry Skip rows and select header to adjust imported table Import format of rows to ship KORI header net included Banking information NETT header Source Plate 10 8 77 Y iD source converted sample s selected sample s CANCEL import Samples Import Samples Delimiter import format Mr of rows to ship header not induded Blanking information EET y header sample Source Bor Drop frame 10 Source Postion temple Position Oropframe ID of blank dest converted sample s selected sample s CANCEL Assign columns to the appropriate description Page 38 A separate blank selection is provided including different blanking options autoblank average of all blanks single blank and multiple blanks Choose the option multiple blanks when diffe
72. is new measurement button on the main menu screen opens the application definition screen to define novel measurements The operating software has been tailored to meet the life scientists needs ement Start new measurement Experiment definition Experiment 2010 10 20 15 52 Sample definition 50 00 ngul Select DropPlate type A and path length Save as Format 25 Task bars CANCEL OPEN TRAY NEXT gt several screen features are built in e Experiment definition The user can define the experiment by name and include extra information in the description box A preset name is generated for every experiment including date and time for easy chronologic storing All the experiment information will be included in the final experiment report e Sample definition Several pre configurated applications have been included for quick and easy experiment definition Detailed description of these applications can be found in chapter 5 o Nucleic acids See chapter 5 1 Unlabeled DNA RNA concentration A260 and purity 260 280 and 260 320 ratio measurements Labeled DNA RNA and fluorescent dye labeling density quantification Page 28 o Purified Proteins See chapter 5 2 Unlabeled protein concentration A280 and purity 260 280 ratio measurements Labeled protein and fluorescent dye labeling density quantification o General UV VIS See chapter 5 3 e Background selection Choice between the defa
73. iven In that case check the quality of the barcode label and try again If the time out warning reappears then contact the dealer for service Bar code read 0111 17 134 100101 Bar code read timeout Time to read 3 25 DropPlate type 15 DropPlate The expiration date is OK 2010 10 29 Time to read 1 05 6 6 Generate status ZIP file This function creates a ZIP file with copy of the internal log files and other information Use this function in case of problems This ZIP file can be send to the dealer for further investigation Create Status Zip file Information Date andtime 2 12 02 A 18 06 2010 CANCEL CREATE STATUS FILE 6 7 Nucleic acid controls several nucleic acid products with an accurate DNA RNA concentration are commercially available These products can be used as routine laboratory control solutions to check the reproducibility and accuracy of the DropSense96 These controls are valid to use with properly calibrated DropSense96 instruments The obtained spectra can be used as an example of what high quality nucleic acid spectra look like and what the expected A260 A280 ratio is of pure nucleic acid samples Page 102
74. length selection When using DropPlate D disposables the user can select between a single or dual path length measurement see 3 1 2 The single measurement requires 1 ul of sample to perform a single pumping step and a small chamber measurement for high concentration sample analysis 2 110 OD 10mm equivalent absorbance The dual measurement requiring 3 ul of sample uses both measurement chambers for the analysis creating the large concentration measurement range 0 05 110 OD 10mm equivalent absorbance When using a DropPlate S only a single measurement is possible This consumable requires 2 ul of sample and performs a single measurement with a measurement range of 0 05 44 OD 10mm equivalent absorbance Save as a format When all the above selections have been made the user can save these in a new format for future use These formats are account dependent Only the lab manager account has the possibility to make a format that is available for all users The formats can be deleted renamed When all sample properties are entered press the next button on the tast bar below Page 67 5 2 3 DropPlate96 sample layout Pressing the next button on the Start new measurement screen will open the next screen where information on sample data and DropPlates96 layout can be entered The positioning information of samples or blank can be entered manually or can be imported from an excel txt or csv file Both import met
75. m in the single well display Optical registration of measurement cycle Empty measurement Transmison measurement Dual path measurement Report display of measurement cycle registration Red Both measurements failed Possible reasons no sample was loaded sample to viscous to load or air bubble entrapment influencing both measurements Orange Only the short path length was used Reasons 1 the single measurement mode was selected 2 not enough sample volume or air bubble entrapment influencing the dual path measurement Black measurement OK both path lengths were measured When using DropPlate D consumables the software will automatically select one of the dual path lenght measurement and normalizes the data to a 1 0 cm 10 0 mm path As a general rule the 1mm pathlenght is used up to OD 1 OD 10 when normalized 1 cm path Above 1 OD at 1 mm the concentration is calculated with the 0 2 mm path absorbance measurement During the measurement cycle the transport of the sample through the microfluidic structure is monitored by continuous spectral measuring This way errors occuring during the process can be detected and displayed on the screen This allows a thourough and robust analysis of all measurements A color legend is used as illustrated on the previous page When using DropPlate S consumables the software will normalize the single cuvette measurement 0 5mm path to a standard 10 0 mm path Simil
76. material Enter the DropPlate of choice DropPlate D or DropPlate S and the target absorbance of the solution given by the manufacturer normalized for a 1mm path length The DropQuant software will automatically calculate the expected OD values for all the path lengths used in the DropPlate Load the blank and potassium dichromate solution on a DropPlate as indicated on the screen It is preferred to use the potassium dichromate solvent as a blank If not present a pure water sample is a good alternative Page 98 Then press the Next button at the bottom of the screen to start the measurement Potasium dichromate test Potassium dichromate test DropPlate D i Potassium Dichromate gf CANCEL When the calibration measurement is finished the 96well display opens giving an overview The column display and single well display can be used for detailed analysis of the spectra GLOBAL crcinean Results RECALC DEBUG REPORT DISPLAY SINGLE WELL oie oe hn 1 j vu a a o A f 4 i L 1A vx LT l un zo oe AUN EISE Fi ts V i 44 1 P 1 A hn n d ah w e i 5 A n un EL ARE ER 4 BACK TO CHANGE DATABASE EXPORT EXFONT PARAMETERS Page 99 In the report display the DropQuant software will show the results of the valida
77. minimized samples maximized throughput DropSense96 User manual Page 1 Revision History Catalog No 10100096 Revision Date December 2010 This manual is published by TRINEAN NV SA Questions or comments regarding the content of this manual can be directed to the address below or to your TRINEAN representative TRINEAN NV SA Dulle Grietlaan 17 3 B 9050 Gentbrugge Belgium Tel 0032 9 2727535 Fax 0032 9 2727539 info trinean com www trinean com DropSense 96 and DropPlate16 96 are trademarks of TRINEAN NV SA L a IIIa aaam This document is the copyright of TRINEAN and must not be copied or reproduced in any form without prior consent TRINEAN reserves the right to make technical improvements to this equipment and documentation without prior notice as part of a continuous program of product development This manual supersedes all previous editions Page 2 Page 3 Contents 1 5 OVERVIEW 6 1 1 PRODUCT DESCRIPTION 6 1 2 INTENDED USE OFTHEINSTRUMEN T oou Di 7 1 3 OVERVIEW TECHNOLOGY E 7 1 4 THEORY OF OPTICAL SYSTEM mrina e i a EEEE 8 1 5 APRICA TON A OAA 9 1 6 COMPONENT 9 1 6 1 9 1 6 2 BOC
78. n ls template Delimiter tab tamale Pa at FP1636 05 3 P1 EP1636 06 3 import format Default converted 5 more Info selected samples select all deselect all DropFrame DropFrame DropFrame Blank zw Position CANCEL Copy pasted excel data Automatic generated table f D _ A 1ST CTCL n Import Samples 010 005408 811 Oropframe 2 8110018257990 DropFrame 2 H12 sample Select delimiter type 010 005408 C11 OropFrame 2 C11 0018858002 DropFrame 2 H12 sample 010 005408 011 Dropframe 2011 0012552014 sample 010 005408 E13 DropFrame 2 11 0012252026 C ample LIX nbl H wre stab LURT z Delimiter to create table space aol 801 1 OropFrame 2 Dropframe2 Import format more info 0 0 deselect all Dropframe ID converted sample s selected sample s select all Source Position Page 36 0018857968 Dropframel 910067909 e sample 0018257980 DropFrame 1 sample general Blank Position DropFrame Sample type CANCEL Alternatively the information in the excel table can be copy drag and dropped directly from the opened excel sheet into the blank window on the import sheet This information will also be used to complete the associated table below When no excel table is available press the open xls t
79. ng your supplier for technical support Page 10 2 Installation 1 Always make sure the power switch on the instrument is in the OFF position and remove the power cord from the back of the instrument prior to any installation or relocation of the DropSense96 2 Do not touch or loosen any screws or parts other than those specifically designated in the instructions Doing so might cause misalignment and voids the instrument warranty 3 Contact your supplier if you experience any difficulties with this instrument 2 1 Unpacking and positioning Remove the instrument from its packaging and inspect it for signs of damage If any are discovered inform your supplier immediately The DropSense96 is packed in a specially designed box Please retain the box and the packing materials If the unit should need to be returned for repair you must use the original packing materials and carton for shipping If the box has been damaged in transit it is particularly important that you retain it for inspection by the carrier in case there has also been damage to the instrument Open the top of the box and remove the packing material from the top and sides of the instrument Lift the instrument up and out of the shipping box Place the instrument down carefully on a stable level surface that can take its weight 25 kg and position it so that air can circulate freely around the casing The accompanying white box contains the power cord USB cable the
80. not take place The software will return to the main menu The progress of a measurement can be observed in the small measurement screen showing wells that are measured indicated by a green color and done wells being measured orange and indicating busy and wells that are still waiting to be measured white color and indicating To do 5 3 5 Measurement e Coinean ABORT TEST Page 88 Data analysis When the full measurement is done following screen appears crinean ion ion io coe aa e fe fe fe ppp 225 ABORT TEST Lae ne Lave Le ne ove fe Please remove disposable from tray You can take the DropPlate96 of the microplate tray and press OK Now the software will generate the full spectral scan of all samples and calculate the concentration of the protein samples For safety reasons the microplate tray with the DropPlate96 will be pulled in automatically after 30 seconds and the measurements will be displayed Several display options can be chosen As default an 96well overview of the measurements is given Type of display for analysis Results Te OD Long OD Short OD Long Short 4A Choice of spectra i 56 zm pru HE ES BACK TO OPEN TRAY DATABASE Page 89 5 3 5 1 96well display Gives an overview of all spectra and the concentration of the nucleic acid calculated f
81. on the PC is required to install the software When attaching the USB cable please wait at least 30 seconds for the USB devices and internal drivers to be installed and recognized 2 2 2 Software installation To properly install the operating software Close all programs and make sure that the USB cable is unplugged Insert the operating software CD in the CD drive of the PC The software installation menu should appear automatically If software menu does not appear choose My Computer to view the contents of the CD Double click on the file named setup exe The first screen will ask the installation path It is recommended to use the default installation path After accepting the License Agreement the DropQuant software will be installed Finally some additional installations will be performed These installations are necessary if you want to make full use of the DropQuant software o PDF Creator is used to create PDF reports o SQLite is used to create a database of past experiments o USB driver is necessary to make connection to the DropSense instrument Page 12 If these additional installations were not performed during initial software installation then they can be installed afterwards from Start gt All Programs gt Trinean DropQuant gt Additional installers e Before you start the DropQuant software power on the instrument and connect the USB cable e Start the DropQuant software by the shortcut on the de
82. onitored by continuous spectral measuring This way errors occuring during the process can be detected and displayed on the screen This allows a thourough and robust analysis of all measurements A color legend is used as illustrated on the previous page When using DropPlate S consumables the software will normalize the single cuvette measurement 0 5mm path to a standard 10 0 mm path Similar color legends are used as with the DropPlate D A black background indicates a good measurement while a red color inducates a failed measurement As the DropPlate S only contains one micro cuvette no orange color legend is used 5 3 5 2 Column display Choice of spectra Choice of data shown Spectrum name and data shown on display under spectrum of sample_4 in well D1 Results REPORT DISPLAY SINGLE WELL DISPLAY COLUMN DISPLAY Spectrum OD Long gli Short v A355 L mm A355 0 2mm A520 L mm A520 0 2mm Column selection BACK TO DATABASE Page 91 5 3 5 3 Selected well for display 96well overview Gives an overview of all measurements per column Clicking on another column opens all spectra of this column Underneath every spectrum the sample name and data are displayed A data selection can be done using the drop down box above the spectra A choice can be made between the OD values of the designated wavelengths The view of the spectra can
83. or display 96well overview Gives an overview of all measurements per column Clicking on another column opens all spectra of this column Underneath every spectrum the sample name and data are displayed A data selection can be done using the scroll button above the spectra A choice can be made between concentration A260 A230 or A260 A280 ratio The view of the spectra can be changed from OD 10mm to the combined view OD long OD short using the Spectrum Type button 5 1 7 3 Single well display Gives a detailed view of a sample spectrum and all additional sample data next to the spectrum A sample can be viewed by clicking on the name of the well or sample in the select input well table or by clicking on a well in the 96well overview Choice of spectra shown on display Results Results 1 REPORT DISPLAY SINGLE WELL DISPLAY Select InputWell OD 10mm sample 1 Ej autoscale sample 2 sample 3 sample 4 sample 5 sample 6 sample 7 sample 8 sample 9 sample 10 sample 11 sample 12 sample 13 sample 14 280 300 320 340 380 Wavelength EXPORT SELECTED TO XLS DATABASE EXPORT hide cursdr show hide cursor with display of wavelenght and OD wavelength nm 260 10mm Absorbance OD 30 96 Concentration F2 1238 52 ng ul Sample Position F2 x information Type sample Autoblank Hame sample 14 Concentration 1238 52
84. ot CANCEL OPEN TRAY ME PREVIOUS START TEST gt PLATE LAYOUT PLATE LAYOUT overview table Select DropPlate96 Addition of phosphorylation information jo Overview OligoWlequences Select disposable DrapPlate 1 Position Sample Name Sequence DNA RNA E MW Conc Factor B9 sample 66 CCTTGGATCCGGGG DNA fiz 127600 4295 8 3367 sample 67 pCCTTGGATCCGGGG DNA A 127000 4374 i ae 09 sample 68 PPPCCTIGGATCCGGGG DNA 127600 4532 7 35 52 Ea sample_69 CCTTGGATCCGGGGp DNA 127600 4374 a 34 29 T F9 sample 70 UUAGCUUGC 84500 27927 3305 EXPORT XL5 Enter sequences Direct calculation Automatic overview of sample M of oligo properties positions and names entered x Export data to excel Page 48 When using the import option section 4 1 4 2 two extra columns need to be selected so the software can import all the oligo sequences from the used data One column containing the sequences need to be selected As for the manual sequuence input phosphorylion of oligonucleotides can be entered using the right number of the letter p added to the 5 or 3 end of the sequence For correct calculation of the extinction coefficient a selection of nucleic acid type of the oligo has to be made by selecting DNA RNA or assignment of a column with that information the column
85. r the meander reservoir is shorter and can include up to 2 5 ul samples Furthermore the DropPlate16 S contains only one micro cuvette with a path length of 0 5mm The 0 05 44 OD range for a 10mm equivalent path of the DropPlate S is ideally suited for quantification of biomolecule samples derived from automated extraction procedures with a stable yield For example extracted human genomic DNA tends to be within a 50 1000 ng ul concentration range and can be quantified using the DropPlate S Since the DropPlate S contains only one micro cuvette instead of two like the DropPlate16 D the measuring time for a full 96well DropPlate S is decreased by halve to about 4 minutes DropPlate S Input well Storage reservoir Micro cuvette Measurement cycle Step 1 Step 2 Step 3 Sample is dispensed into Sample is drawn into the In the DropSense96 the the input well manually or by reservoir by capillary action micro cuvette is filled using a a liquid dispensing robot for storage and sample small vacuum pressure An protection absorption measurement is performed Sample size requirements Although the dispensed sample volume is not critical it is essential that a minimal amount of sample is dispensed for correct filling of the measurement chamber allowing precise measurements Extensive testing indicates that sample volumes of 2 yl is sufficient to ensure reproducibility Although this volume range takes a pipetting error into Pa
86. rent blanking solutions are used for separate sample groups in the experiment In that case two extra columns need to be present in the imported file one column shows the DropPlate ID and the other the well position of the blank on that DropPlate An example can be seen by opening the open xls template button When only one blank is used for the complete experiment then choose the single blank option and type in the blank well position and the specific DropPlate ID on which the blank is loaded or type in the name of the blank software will locate the correct position from the name When a single blanking solution is loaded more than once and the average of those measurements should be used as blanking information then select the average of all blanks and type in the blank name The same name must be used for all the loaded blank wells so that the software can locate them all and determine the average of all these blanks Note The ID of the DropPlate containing a single blank can be entered or an external bar code reader can be used to read the bar code on the side of the DropPlate Import Samples Import Samples open ls template Delimiter Ure source weli semple 5 i d jaa eoa zm a 3 I f Selection of 1636053 Pa ER1636063 blank type suyteblanl format D
87. rom A260 See example above In the standard mode all spectra are shown normalized to a 10mm path For a DropPlate D measurement a combined spectral view of both path lengths can be shown by selecting OD long OD short in the Spectral Type button Detailed analysis of one sample can be achieved by clicking on the sample spectrum opening a detailed figure of this spectrum in the single well display Optical registration of measurement cycle Empty measurement Transmison measurement Dual path measurement Report display of measurement cycle registration Red Both measurements failed Possible reasons no sample was loaded sample to viscous to load or air bubble entrapment influencing both measurements Orange Only the short path length was used Reasons 1 the single measurement mode was selected 2 not enough sample volume or air bubble entrapment influencing the dual path measurement Black measurement OK both path lengths were measured When using DropPlate D consumables the software will automatically select one of the dual path lenght measurement and normalizes the data to a 1 0 cm 10 0 mm path As a general rule the 1mm pathlenght is used up to OD 1 OD 10 when normalized 1 cm path Above 1 OD at 1 mm the concentration is calculated with the 0 2 mm path absorbance measurement During the measurement cycle the transport of the sample through the microfluidic structure is m
88. rotein sequence of the protein to be measured is known the theoretical molar extinction coefficient can be calculated using the equation 5500 Trp 1490 T yr 125 Cys Where the extinction coefficient number of Trp Tryptophan Tyr Tyrosines Cys Cysteines A very rough protein concentration can be obtained by making the assumption that the protein sample has an extinction coefficient of 1 so 1 OD 1 mg ml protein In combination with the DropPlate D consumable the DropSense96 performs a dual path measurement with path lengths of 0 2 and 1mm to enable quantification of proteins with a wide concentration range without need for dilution Using the DropPlate S with a single micro cuvette 0 5mm path on the DropSense96 is ideal for quick analysis of protein samples within a more limited measurement range The absorbance data are archived in the database and displayed on the software screen after measurement In the protein A280 application the software chooses automatically which path length generates the best absorbance values and normalizes this measurement to a 1 0 cm 10 0 mm path These are displayed in the software for further data analysis Page 62 Since the UV absorption of nucleic acids at 280 nm can be as much as 10 times that of a protein a small percent of nucleic acids in the sample can greatly distort the protein quantification Therefore the protein sample purity must be determine
89. s ready but no communication with the PC has been achieved check USB connection 3 Green light DropSense96 is ready and connected with the PC 4 Green and orange light busy with measurement 5 Red light error state see troubleshooting or contact your distributor 9 Microplate drawer The microplate drawer slides in and out of the microplate chamber that is covered by a lid on the front panel The drawer remains in the reading chamber during measurement cycles The drawer can accommodate 1 DropPlates16 which are placed on a dedicated aluminum DropFrame 2 full disposable DropPlates96 and 3 standard 96well microtiter plates The drawer is specifically designed to be compatible with robotic handling The plate carrier can be opened at any given time using the open tray button in the DropQuant software Do not obstruct the movement of the tray 1 6 2 Back panel The following components are located on the back panel of the 96 1 Power switch a rocker switch labeled I O for on and off respectively 2 Power cord receptacle plug the power cord in here 3 Free computer USB port Plug the accompanying USB cable into the USB port of the DropSense96 attach the other end to a USB port of the computer 4 Label provides information about this DropSense such as line voltage rating cautionary information serial number Record the serial number shown on this label for use when contacti
90. significant protein phenol or other aromatic compound contamination However the A260 280 ratio is not always an accurate representation of DNA purity Other contaminating substances like EDTA and carbohydrates have a low 280 nm absorption but absorb UV light around 230nm Furthermore some proteins containing few aromatic residues have little absorbance at 280 nm while all proteins have a clear absorption peak at 228 nm due to their peptide bonds This makes the 260 230 ratio often a more constant indicator of the presence of protein in a nucleic acid sample Therefore absorbance readings measured both at 230 nm and at 280 nm provide a more accurate estimate of contaminants that may be present in nucleic acid samples The ratio of the A260 A230 should be 1 8 or greater since nucleic acids have an absorbance minima at 230 nm lt is recommended to use the absorbance readings at A260 280 and 230 and examining both A260 280 and A260 230 ratios for every sample As a general rule a 260 280 ratio of 1 8 and a 260 230 ratio of 2 0 or greater predict clean DNA good quality RNA will have a 260 280 ratio of 1 8 2 0 and an OD ratio 260 230 of 2 0 or greater The 260 280 ratio can differ depending on the spectrophotometer used It is dependent on both the characteristics of the sample pH ionic strength and the wavelength accuracy of the spectrophotometer used Since the DNA absorption peak shows a steep slope at 280 nm a slight shi
91. sktop or go to Start gt All Programs gt Trinean DropQuant gt DropQuant 2 2 3 Registering Your Instrument Please register your instrument We periodically update our software and add new features free of charge We keep our user list updated so that we can alert you to these updates information supplied is completely confidential Please register your instrument on our website www trinean com user register or mail to info trinean com 2 2 4 Setting up the instrument Install your DropSense96 instrument as follows e Place the DropSense96 on a stable level surface that can take its weight 25 kg away from direct sunlight dust drafts vibration and moisture Position it so that air can circulate freely around the casing e Turn the DropSense96 around so that the back is facing you and insert the USB cable into the USB port receptacle Attach the other end to the computer Connect the instrument to the power supply with the power adaptor supplied e Turn on the power to start the instrument rocker switch at the back Start up the software so that a connection can be established Caution Strong light source Never look directly into the beam of any UV Visible spectrophotometer The instrument is fitted with safety interlocks If the instrument is used in a manner not specified or in environmental conditions not appropriate for safe operation the protection provided may be impaired and instrument warranty withdra
92. ssing Results Post Processing Results REPORT DISPLAY SINGLE WELL DISPLAY Choice of spectra shown on display BACK TO DATABASE TRAY 5 1 7 1 96well display Gives an overview of all spectra and the concentration of the nucleic acid calculated from A260 See example above In the standard mode all spectra are shown normalized to a 10mm path For a DropPlate D measurement a combined spectral view of both path lengths can be shown by selecting OD long OD short in the Spectral Type button Detailed analysis of one sample can be achieved by clicking on the sample spectrum opening a detailed figure of this spectrum in the single well display Optical registration of measurement cycle Empty measurement Transmison measurement Dual path measurement Report display of measurement cycle registration Red Both measurements failed Possible reasons no sample was loaded sample to viscous to load or air bubble entrapment influencing both measurements Orange Only the short path length was used Reasons 1 the single measurement mode was selected 2 not enough sample volume or air bubble entrapment influencing the dual path measurement Black measurement OK both path lengths were measured When using DropPlate D consumables the software will automatically select one of the dual path lenght measurement and normalizes the data to a 1 0 cm 10 0 mm path As a general rul
93. t the report option All absorbance values 10mm will show all the absorbance values within the selected wavelength range of the X axis So manual changing of the X axis scale will change the info in the report e Blank subtraction When using a blank reference sample it is automatically subtracted from the sample absorbance before concentration calculation This feature can be turned off so the spectral shape and concentration calculation is shown without blank compensation In this mode the shape of the blank spectrum itself can be analyzed When the blank subtraction is turned off all other screens and the report section will generate data without blank subtraction e Background correction the background correction method can be selected in the start new measurement sheet section 5 1 2 The background information is derived from the RAW OD spectrum measured In the default setting a linear fit through the 400 600 nm region of the transmission spectrum is subtracted from the full spectrum while in the single point mode the background level of a wavelength of choice is subtracted from the full spectrum The manual selection on the single well display offers the option to chose between the default method a single wavelength of choice or no background correction This allows to verify if the background is not near zero as indicator for turbidity or presence of debris Note that changes in the background Page 93 corr
94. ted columns iic as Source Plate ID DropPlate ID Einclude Summary Source Position DropPlate Position El include 96 weil plate layout Export file type Sample name selection Date Pump xls __openin existing Excel file Time gt 4957 0 2mm OD e pat Constant c 2257 1 0mm 00 Instrument ID A350 0 2mm OD direct print Path length mode A350 1 0mm OD All Absorbance Values 0 2mm z All Absorbance Values 1mm csv Preview table Preview DropPiate Samplename Pump 257 A257 A350 Portion 2mm 1 0mm D 2mm aD ioni jon DropFiate 1 Al bam 1 ooo ooo 0000 Droplatel A2 sample 9 018 01 Create report DropPiate 1 82 sample 10 0124 0 614 0 003 DropFlata1 ca sampla 11 0185 0130 DropPiatel 02 sample 12 014 om DraopPiata 1 _ 17 0 186 0 922 0 130 1 outliers EXPORT RESULTS save save as rename Save as format Page 96 6 Diagnostics 6 1 System Info and Settings The diagnostic tools are only available for a log in with an entry level as lab manager A extra software button is located on the left bottom side main screen Systems Info and Settings DropQuant DropQuant for DropSense 96 mmini l in System Info and Settings System Info Edit Settings Computer Diagnostics Operating System Microsoft W
95. ted from the full spectrum The manual selection on the single well display offers the option to chose between the default method a single wavelength of choice or no background correction This allows to verify if the background is not near zero as indicator for turbidity or presence of debris Note that changes in the background correction will be used in the other screens and report section In the report section the RAW OD value of the wavelength shown in the single point background correction for instance RAW A405 10 mm can be included in the report table to export The single well display offers a quick export to XLS function located underneath the spectral view This function is limited to an export to Excel showing all the spectra selected on the single well display and a fixed selection of columns with all necessary data from the selected samples An example is shown on the next page Page 79 e DropSense REPORT 10mm Absorbance OD ee E BEE eee T mH L ET E m L1 1 1 4 LI E E T LA Ad AOOO l 230 240 260 280 300 320 340 360 380 400 Wavelength DropPlate E Concentration A260 A280 Raw A405 DropPlate ID e Sample name 5 A260 A280 Position
96. the DropPlate S Single and DropPlate D Dual Both have identical conical shaped input wells connected with a meander shaped storage reservoir that uses capillary force to take up the sample from the input well and protect the small samples from evaporation The other end of the meander reservoir is connected to one DropPlate S or two micro cuvettes DropPlate D for UV VIS analysis The choice of DropPlate16 to be used depends on the desired OD measurement range of the experiment and measurement speed as explained below 3 1 2 1 The DropPlate D The DropPlate D Dual contains a meander shaped reservoir that can store up to 3 5 ul of sample and two superposed micro cuvettes for optical analysis By consecutive filling of both optical chambers a dual path length measurement is performed 0 2mm and 1 0mm The combination of both path lengths results in a large OD measurement range and thereby omits the need for sample dilution Page 15 DropPlate D Input well Storage reservoir Micro cuvette Measurement cycle Step 1 Step 2 Step 3 Step 4 Sample is dispensed into Sample is drawn into the In the DropSense96 the first The second micro cuvette is the input well manually or by reservoir by capillary action micro cuvette is filled using a by a higher vacuum liquid dispensing robot for storage and sample small vacuum pressure An pressure An absorption protection absorption measurement is measurement is performed performe
97. the DropPlate96 frame Page 18 3 1 3 2 Manual sample loading on a DropPlate Take care not to introduce large air bubbles into the dispensed sample When dispensing samples into the DropPlate put the tip of the pipettor into the input well at an angle of 10 angle or more until the tip makes contact with the inner wall of the well and then deliver the liquid by gently pressing the push button of the pipettor until the first stop pressing further down to the second stop inevitably will create bubbles in the sample Lift the pipette up carefully after dispensing The sample will be sucked automatically into the meander reservoir by capillary force The black colored aluminum DropFrame and 96well mould of the DropPlate96 create a difference in visual contrast between empty and filled meander shaped reservoirs of the DropPlate disposables This allows a quick and easy visual control of the loaded wells Furthermore this visual inspection can be used to estimate the amount of sample dispensed or the presents of air bubbles in the samples as shown in the figure below The dispensed sample will automatically move into the channel reservoir by capillary force Therefore the sample does not need to be pushed into the reservoir by the pipettor Pg DropPlate D 4 1 3 pl rai 2 2 5 ul 32 L2 a DENT R 4 1 5ul men a 4 o empty Page 19 3 1 3 3 Automated sample loading on a DropPlate
98. the measurement is within the expected measurement variation The DropSense96 performs within specifications e Red color indicates that the measurement does not fall within the expected variation Contact your local distributor for a systems check up A report txt xls be created using the export button This report can be stored on a disk for future reference 6 4 Quick system check This tool performs a quick check of the spectrophotometer module and the pump module in the DropSense96 This quick check starts automatically when activated and takes about 30 seconds The Quick system check consists of e Opectrophotometer check the lamp spectrum is measured and compared to the lamp spectrum at calibration The report summarizes the measured amplitude and position of the wavelength peaks and gives a warning if these parameters deviate too much Page 100 from the calibration values This indicates that the lamp or spectrophotometer may need service e Pump system check the output pressure of the pumps is measured using the internal pressure sensors and compared to the values at the calibration This allows to check for degradation of the pumps e The operation of the internal background light sensor and the internal temperature sensor is checked Spectrum Stability Reference Pixel Position 0 168 in range max 2 000 Reference Pixel Amplitude 0 009 in range max 1 000 Reference Pixel
99. tion procedure All OD values measured by the 4 optical systems the DropSense96 are shown Results SINGLE WELL DISPLAY COLUMN DISPLAY CHANNEL 1 MEASUREMENT DATE TEST PERFORMED BY 21 01 2010 10 50 28 trinean INSTRUMENT ID PATH LENGTH 2005 002 Double path length EXPERIMENT Experiment 2010 21 01 10h 49 CHANNEL 2 INFORMATION Test new CF 1 calibration method on D52003 07 SOLVENT water based solvents ind TE 5 AT buffer MATERIAL Potassium Dichromate Target Absorbance 1 0mm pathlength 0 742 OD Target Absorbance 0 2mm pathlength 0 148 OD CHANNEL 3 CHANNEL 4 CALIBRATION WITHIN SPECIFICATION S BACK TO DATABASE EXPORT DATA A350 1 0mm 4450 0 2mm 0 735 0 145 0 739 OD 0 151 OD 0 74100 0 147 00 0 74000 0 14900 A330 L mm A330 0 2mm 0 74000 0 14900 0 74400 0 15200 0 73900 0 14900 p 737 OD 0 149 A350 LOmm 250 0 2mm 0 742 OD 0 152 OD 074000 0 15300 0 7400D 0 14800 0 744 0 150 OD A350 L0mm A350 0 2mm 0 713 0 137 0D 0 72700 0 14700 0 731 OD 0 147 OD EXPORT GLOBAL gt RECALC DEBUG AVG Terror AWG error Ela CHANGE PARAMETERS For every optical system the mean of all measurements are calculated and compared with the expected OD value of the potassium dichromate solution Color indications are foreseen giving a clear overview e green color indicates that
100. to Detailed description of the PC configuration o Detailed info on the DropSense96 o Diagnostic checks further explained in chapter 6 o A general status of the Dropsense96 can be zipped and sent to the dealer for a first system analysis Settings system Info and Settings System Info Edit Settings Settings t i System Info and Settings Users See Edit Settings Meaiurement Database Location Computer Diagnostics Operatinq System Microtoft Windows XP Service Pack 3 5 1 Potatium dichromate test a4 111 version 04 11 41 Quick system check Formats PIN Creator version jos Export Import SQLite installed heck t Export Settings Drop Quamt Memote veriten installed Disk Status Import Settings Total divhapace 252056 Generate Status Zip File Free diskipace MB 1154275 Drop 5ense Oropiense Serial Number 2005 001 i bor hema 146 internal bar code reader nonr Nr Pulses Lamp 692945 Last Calibration 7 6 2010 Nr Pumpcycies 679 OPEN TRAY firmware version d S Nr Pumpcycles pumpi 662 Pump Tone 5 pumpo 6315 Pump Time 15 pump 5505 OPEN TRAY Page 25 Edit settings o Edit Users Add or change user accounts and passwords At the lab manager level the password can be personalized During installation a random lab manager password has been given which can be foun
101. tra selected on the single well display and a fixed selection of columns with all necessary data from the selected samples An example is shown on the next page Page 58 e DropSense REPORT 33 10mm Absorbance OD I I I 230 240 260 280 300 320 340 360 380 400 Wavelength DropPlate Concentration A230 A260 A280 Raw A405 DropPlate1 G1 sample 7 124089 1342 3102 1554 0 04 231 2 DropPlate1 H1 sample 8 109233 1206 27 31 1426 007 226 DropPlate1 A2 sample9 114849 1239 2871 1447 037 232 DropPlate 1 B2 sample 10 12064 1301 3016 1511 005 232 2 DropPlate1 C2 sample 11 186 025 0 47 033 0 06 188 142 sample 12 2107 029 053 035 006 183 15 sample 13 2278 026 057 038 008 218 15 3011 031 075 057 015 24 132 7892 086 197 142 005 23 139 sample 18 106232 1442 2656 1305 06 184 204 DropPlate 1 sample 19 117011 149 2925 1388 169 19 211 Do E LE ee 2 DropPlate 1 G2 sample 15 3837 041 0 0 67 013 236 143 E E Page 59 5 1 7 4 Report display The report section gives an overview of the experiment information and all the measurement data in a table format The info on the data table can be altered by clicking on the header
102. tune the pressures used by the vacuum pumps guaranteeing precise and controlled filling of the microfluidic structures All water based solvents containing low amounts of salts and buffers are grouped in one option since these substances don t affect the pumping properties waler based solvents inc TE 5 RT buffer water glycerol 5 10 water DMSO 5 10 Choice solvent Extra button for high viscous samples An extra button for high viscous samples can be used so the pumps will function at higher pressure and longer duration to fill both measuring cuvettes on the DropPlates correctly Page 84 Viscous samples should be handled with care as high viscosity can hamper both precise pipetting of the samples and transport of the sample in the microfluidic DropPlate16 When the sample is still too viscous the dual measurement can fail This will be indicated by an orange or red color indication when the results are shown see further Furthermore it is prohibited to use other solvents then described in the DropQuant software Extra caution should be taken with samples containing detergents as they may have more hydrophobic properties hampering controlled filling of the microfluidic structures Background correction Sample Sample Type Nucleic Acids Purified Proteins ele ees 1 Sample Material dsDNA Solvent water based solvents incl TE PBS RT buffer concentration fa
103. ult background correction using the spectral range of 400 600 nm or a single wavelength of choice e DropPlate type Depending on the application or sample concentration range a choice of DropPlate S or DropPlate D can be made Both DropPlates are explained in detail in section 3 1 Alternatively a standard 96well microtiter plate can be read This selection requires additional information like path length or sample volume and plate brand e Path length selection When selecting the DropPlate D the user can further choose between a single or dual path length measurement The single measurement requires 1 pl of sample to perform a single pumping step and a small chamber measurement for high concentration sample analysis 2 to 110 OD for a 10mm path The dual measurement requiring 3 ul of sample uses both measurement chambers for the analysis creating the large concentration measurement range 0 05 to 110 OD for a 10mm path When using a DropPlate S only a single path length measurement is possible This selection requires a sample volume of 2 ul and generates a measurement range of 0 05 44 OD for a 10mm path e Save as Format When all the above selections have been made the user can save these in a new format for future use These formats are account dependent Only the lab manager account has the possibility to make a format that is available for all users The formats can be deleted renamed e Task bars A general task bar is included
104. und wavelength of choice will be zero During the data analysis the background correction can be altered again or turned off for detailed analysis Sample Sample Type Nucleic Acids Purified Proteins General UV vis bee Sample Material dsDNA Solvent water based solvents incl TE 5 RT buffer concentration factor 26 nm 260 High viscosity concentration units ng ul Background correction default single point 1340 nm Background correction options Page 66 DropPlate selection Depending on the application or sample concentration range a choice of DropPlate D or DropPlate S can be made Both DropPlates are explained in detail in section 3 1 Alternatively a standard 96well microtiter plate can be used This selection requires additional information like path length or sample volume combined with plate brand and type in order to calculate the correct path length Additional plate brands and types can be entered by selecting Create new from the drop down list Selection of path length or volume DropPlate Type DropPlate type Standard 96 well plate path length lt sample volume 200 0 path length E wellplatename EAE EE Corning 96 ell UV plate Greiner 96 well UV Star 5 Thermo Microtiter 96 ell plate as rename delete 96 ell NUNC plate Create new Selection of brand and type of 96well plate Path
105. urned off for detailed analysis Page 44 DropPlate selection Default DropPlate selection Path length selection Save as format Depending on the application or sample concentration range a choice of DropPlate D or DropPlate S can be made Both DropPlates are explained in detail in section 3 1 Alternatively a standard 96well microtiter plate can be used This selection requires additional information like path length or sample volume combined with plate brand and type in order to calculate the correct path length Additional plate brands and types can be entered by selecting lt Create new gt from the drop down list Selection of path length gt orvolume Standard s6wellpiate C KD 21 Z Falcon 96 well UV plate Corning 96 vell UY plate Greiner 96 vell Uv Star 6 Thermo Microtiter 96 1 plate 96 vell NUNC plate Selection of brand and C Create new type of 96well plate Path length selection When using DropPlate D disposables the user can select between a single or dual path length measurement see 3 1 2 The single measurement requires 1 ul of sample to perform a single pumping step and a small chamber measurement for high concentration sample analysis 100 5500 ng ul dsDNA The dual measurement requiring 3 ul of sample uses both measurement chambers for the analysis creating the large concentration measurement range 5 5500 ng l dsDNA When using a DropPlate S only a single m
106. ven by the manufacturer like the appropriate correction Page 50 factors can be entered The 260 nm correction will be utilized for nucleic acid concentration calculation when the respective dye is selected To remove a dye select the dye and press the Remove label button Pre defined dyes identified by a next to the label name may not be edited or deleted Labels Labels Database 280nm correction l Extinction Coeff Absorbance wavelength 260nm correction 250000 Alexa Fluor 488 71000 Alexa Fluor 546 104000 Alexa Fluor 555 150000 Alexa Fluor 594 73000 590 Alexa Fluor 647 239000 650 0 00 0 03 Alexa Fluor 660 132000 663 0 00 0 01 Cy3 5 150000 581 0 00 0 00 cy5 5 250000 675 0 00 0 00 DyLight 488 70000 493 684 0 03 0 11 0 21 0 04 0 08 e c o e e e ln ccc amp e un a t 0 23 0 15 Add Label Remove Labe im a Save Changes Press the Save changes button and then Back to return to the New measurement page Page 51 5 1 6 Start measurement Press the Start Test button on the task bar of the Plate layout screen This will open the microplate tray of the connected DropSense96 and the following screen will appear 1 2 3 4 5 T 8 9 10 12 gt O 1 1 1 1 1 4 1 4 1 1 1 ABORT TEST Please insert disposable 1 DropPlate 1 in the tray and press O
107. w Please contact your distributor for a thorough system check and recalibration Warning Warning has a decrease of pressure of 9 1 Reference Pixel Position 2 324 8 0 to 8 0 gt out of range out of range max 2 000 Reference Pixel Amplitude 0 001 1 has a decrease of pressure of 3 1 in range max 1 000 8 0 to 8 0 gt range Reference Pixel Dark 0 004 in range max 0 160 3 2 2 Loading of a DropPlate96 on the DropSense96 The plate carrier of the DropSense96 can be opened at any time except during measurements using the open close tray button in the DropQuant software The tray opens automatically when pushing the start test button in the software see further to load the DropPlate96 or a standard 96well microtiter plate before reading When reading is complete the drawer of the DropSense96 opens allowing you to remove the DropPlate The drawer closes automatically after 30 seconds this feature is not active when the DropSense96 is integrated Page 21 The DropPlate must be loaded onto the microplate drawer of the DropSense96 with the well A1 matching the upper left position of the drawer An orientation switch in the upper left corner can be used to avoid incorrect placement of the frame on the drawer Make sure that the DropPlate96 frame is placed accurately and flat on the drawer orientation switch not operational orientation switch operational
108. wn There are no user serviceable parts inside this instrument Page 13 3 Operations 3 1 Sample loading on the Trinean DropPlates 3 1 1 DropPlate General description The Trinean DropPlate consumables have been designed for specific use with the DropSense96 and are made of special optical quality plastic allowing transmission of UV VIS spectra The conical shaped input wells are positioned with a 9mm SBS standard pitch fit for multi channel pipettors or robotic sample loading These input wells are connected with a capillary storage channel and one or two micro cuvettes as shown in the figure below The microfluidic structures continue with a small channel leading to a small upper vent used as the inlet for the vacuum pressure system When dispensing a sample droplet into the input well it is instantly drawn into the storage channel through capillary forces in order to strongly suppress sample evaporation This allows the user to perform the measurement on the DropSense96 within a time span of 2h after dispensing After inserting the DropPlate into the DropSense96 pressure driven transport of the samples to the micro cuvettes occurs while simultaneous absorbance measurements are performed to monitor the filling behavior of the micro cuvettes and analyze the spectral absorbance of the sample The microfluidic chip provides steady measuring conditions due to fixed path lengths and elimination of solvent evaporation leading to enhance
109. yout screen proceed as described in section 5 1 3 and then press the Enter check sequences button Now a table will appear with a overview of the selected wells Type in the proper oligonucleotide sequences or copy past them from an excel table The software will calculate the nessecary properties like the exctinction coefficient the molecular weight and the concentration factor All the information can be saved in excel by pressing the export excel button at the bottom of the screen Phosporylations at both the 5 and 3 end can also be included by adding the right number of the letter p to the sequence Press Save to return to the Plate layout screen When using the Replicates function see section 4 1 4 1 entering the sequence of one sample will automatically lead to the fill in of the sequence and data its replicates Page 47 Manual blank and sample positioning Plate Layout e an Plate AMORE B EH seme compart 1 som VIS seme s N merca men mnes 1 ELE j o f mores memos J sample 25 23 midi nume sample M semplaat a
110. ype of blanking is used for this sample or group of samples A source plate location per sample can also be entered here A convenient way of fast well identification of a sample group is by selecting an area on the DropPlate96 by dragging with the computer mouse Alternatively one by one can be selected by clicking on the correct position on the 96well plate All selected wells will contain the information shown on the left side sample blank empty choice of blank The name of these defined wells can be added to the sample in the box next to Well name or by entering the name in the matching well space in the table at the bottom of the screen by double clicking on the well space As default samples will be given numbered names starting with sample 1 in A1 to sample_96 in well H12 An overview of all well names is given in the table at the bottom of the screen Sample or sample group information select click m ode Position Type well Sample Well name sample Blanking information Autoblank E Source Plate ID Source Position 33 Additional options Using the right PC mouse button on the 96well layout figure will open additional plate layout features to fill in full rows colums or a full 96well plate and delete the plate layout Extra plate layout features using gt the PC mouse a s 8 8 E 8 8 i il 8 8

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