Avans UVS-99 User Manual-EN
Contents
1. UVS 99 UVISDrop Micro volume UV Vis Spectrophotometer USER MANUAL SOFTWARE VERSION UVS 99 V1 1 Contents EOE 1710 10 EE RE EE EO daa tala i a 1 at OCU CMON v P r RO 1 1 2 0p rationa EE N ee a EE ee 2 1 3 Applications ME ER ER N 2 NS AN OS n EE EE NE 3 2 1 Computer requirements EE EE EE ER ee Ee 3 2 2 Software instala HON EE EE EN ER ER EG Ee Re 3 2 9 PIGCAaUNONS ER OE aie 5 General Operation asse EER AE Re Ee de Ge ee 7 3 1 Sample size recommendation session 7 3 2 General measurement protocol 7 3 3 Factors of affecting the measurement results 9 M asurement nn aitu 11 4A Mielie AGO ee sn 11 4 Protein A ARR conan amandes 13 4 9 Cell GUMEDE GE Ge RE Ne Ge RE KEN eme 17 V VVENVIS onda bon anatomie 19 4 5 Protein ECS nc ss np 23 4 6 Protein Bradford aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaannnnnnanannnnnnnnnnnnnnnannaaaa 25 Check and Adjustment of Light Intensity 2aaaaara ri 27 5 1 iese od AE AE N 27 5 4 OCI ANON ss EES EE EI GE RR GE ER Ee DE OE GR RD RE Ge Ge Ee De 27 5 3 Light intensity EBOEK EER DE EE se Ee ee ee Es EE Ee 29 5 4 Light intensity adjustment sesse ee ee Re ee ee ee ee ee ee 30 5 5 Special application AA Gee nn 31 5 6 Set up of the Calibration Parameters 34 Be SA OE N OR N N Oe an dune 34 TEOUDIESPOOIMIAE2. Autosave The system will record and autosave the measurements The maximum number of records is 1000 Save Graph Click Save Graph to save your graph if necessary Report Click Report and a dialog box will pop up Figure 4 6 Press Yes and the full spectrum between A1 and A2 will be exported to an Excel file with 1 nm increment When No button is pressed only the absorbencies of A1 and A2 will be exported to an Excel file Calculation of Sample Concentration There is one function in the UV VIS module which can allow the user to calculate other samples concentration value with the same sample type by setting up and plotting the concentration curve of the known samples As shown by Figure 4 7 click the upper left icon standard then Figure 4 8is seen 20 different Save Grape Record tut P Button Disable O immed O 0 2mm Yellow 20 20 xo mw 40 w s w 650 700 750 BO Wavelengthine Max Absorbance 10 Wavelengtht 350 Abs 014 Sample ID Wavelength2 600 Abs 003 Figure4 7 Standard X Abs Concentration M stat F2 ES eus l sta3 staa r stas Save Graph set Scale l stae star OK Concentration 0 Concentration 5 0 Figure 4 8 In the left hand side of Figure 4 8 there are seven rows for the users to key in absorbance and concentrati
3. sie GE a de 37 6 1 Unable to find USB device iii 37 6 2 Sample accuracy and reproducibility 22aaaannasss nnsnnananssssnnaaaa 38 6 3 Unusual spectrum iaaaaaaaaanaa aaaaaaannnannnnannnnannnnnannnnannnnaanaaaanaaaaa 39 Maintenance and Warranty 40 vale yy EER OE N EE EO EE EE LE n 40 7 2 Parts that require replacements aaaaaaaaaaaaanas s aaaaaaaanasssnaaaaa 40 7 3 Calibration of Wavelength 40 7 4 Solvent Compatibility ses EES BR EE Ee ER BERE EDE Ee Ee ED 41 7 5 Decontamination of Measurement amp Optical Surfaces 41 ele ele AE nn EE 45 8 1 Instrument SPECIFICATIONS aaaaaaaaannasnnnananannnnnannnnnnnnnannanannanaaa 45 8 2 Absorbance calculation sesse ses ke Hi RE Ee EE Ee DE ER RO Eg 46 8 3 Concentration Calculation Beer Lambert Law 46 8 4 Fluorescent Dyes Measurement 47 1 Overview 1 1 Introduction UVS 99 is a full spectrum spectrophotometer 200 850nm that measures 1ul sample with high accuracy and reproducibility It utilizes a new sample retention technology that employs surface tension alone to hold the sample in place No cuvettes or capillaries or other positioning instruments required It s simple to operate and easy to clean by swiping with a laboratory wipe Furthermore UVS 99 can measure high concentration samples without dilution 50xhi
4. 2 osa Sample Type BSA 260Abs _ 1 585 _ 1 585 UVS 99 mm EE Le Figure 4 3 Type the Sample ID and select the Sample Type Sample ID Used to define a name of sample Sample Type Used to select the type of protein being measured 1 Abs 1 mg ml A general reference setting based on a 0 1 1 mg ml protein solution producing an Absorbance at 280 nm of 1 0 A where the path length is 10 mm 14 BSA Bovine Serum Albumin reference Unknown sample protein concentrations are calculated using the mass extinction coefficient of 6 7 at 280 nm for a 1 10 mg ml BSA solution IgG IgG reference Unknown sample protein concentrations are calculated using the mass extinction coefficient of 13 7 at 280 nm for a 1 10 mg ml IgG solution Lysozyme Lysozyme reference Unknown sample protein concentrations are calculated using the mass extinction coefficient of 26 4 at 280 nm fora 1 10 mg ml Lysozyme solution Other User entered mass extinction coefficient L gm em for a 1 10 mg ml solution of the respective reference protein nm Abs User selected wavelength and corresponding absorbance The wavelength can be selected by typing in or using the up down arrows to the left of the wavelength box Note The user selected wavelength and absorbance are not utilized in any calculations A280 Sample absorbance at 280 nm represented as if measured with a conventional 10 mm path A
5. 8 2 2 If the ratio is appreciably lower this may indicate the presence of co purified contaminants Concentration ng ul dsDNA concentration 260 nm Abs x 50 242 SSDNA concentration 260 nm Abs x 33 RNA concentration 260 nm Abs x 40 Other concentration 260 nm Abs x Constant Autosave The system will record and autosave the measurements The maximum number of records is 1000 Save Graph Click Save Graph to save your graph if necessary Report Click Report All results have been recorded will be exported to an Excel file Absorbance data shown in files are represented as displayed on the software screen B EEG D E G _ H SamleID _lsbs260 Abs280 Abs230 260 280 260 230 Concentration ng ul Smaple Type mal 38 398 30 221 19 594 if 1 96 1267 1 ssDNA 3 a2 49 978 30 676 20 662 1 63 2 42 1649 3 ssDNA 4 Jas 40 868 26 494 20 138 1 54 2 03 1348 7 ssDNA 5 ad 46 667 33 087 19 822 1 41 2 35 1540 ssDNA 6 a5 47 0991 30 923 19 262 1 52 2 45 1554 3 ssDNA 7 a6 55 328 31 324 22 144 Lat 2 5 1825 8 ssDNA 8 la7 49 239 31 011 21 201 159 2 32 1624 9 ssDNA 9 jag 60 06 38 688 22 918 1 55 2 62 1982 ssDNA 10 a9 50 496 34 999 22 392 1 44 2 26 1666 4 ssDNA 11 al0 57 675 40 502 25 819 1 42 2 23 1903 3 ssDNA 12 all 43 586 38 866 24 404 1 25 1 99 1603 3 ssDNA 13 al2 55 8 40 094 26 203 1 39 2 18 1841 4 ssDNA 14 bl 44 969 33 88
6. the Products any post installation realignment readjustment re cleaning or recalibration unless due to defects in material or workmanship are the responsibility of Customer Certain items which are refurbished or otherwise reconditioned are also excluded from the terms of this warranty This warranty does not cover any damage due to accidents vandalism fire flood other environmental factors or any other Force Majeure If you have questions regarding warranty coverage please contact your local supplier The warranty further excludes items supplied by third party manufacturers such as computers and related software Customer is responsible for any modifications to the computers or software including but not limited to interfacing or networking unauthorized hardware modifications repartitioning or reformatting of the hard drive installation or modification of additional or new hardware and installation or reinstallation of new or existing computer operating systems or service packs THIS WARRANTY IS MADE IN LIEU OF ALL OTHER WARRANTIES EXPRESS OR IMPLIED INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR 42 PURPOSE ANY IMPLIED WARRANTY ARISING OUT OF A COURSE OF DEALING OR OF PERFORMANCE CUSTOM OR USAGE OF TRADE OR ANY IMPLIED WARRANTY AGAINST PATENT INFRINGEMENT UNDER NO CIRCUMSTANCES SHALL THE MANUFACTURER BE LIABLE FOR DAMAGES OF ANY KIND INCLUDING WITHOUT LI
7. the original K parameter is 0 9 then the new K parameter should be 0 9 100 80 1 125 36 6 Troubleshooting 6 1 Unable to find USB device Error USB2000 Unable To find Device with Serial Figure 6 1 This error might appear upon software startup and usually indicates that the USB cable is not properly connected or the software is not installed properly Please follow the following steps to double check eCheck the USB cable and ensure that it is plugged into both the computer and the UVS 99 e Check the power adaptor Please check if the light on the adaptor is on e Check if the connection of the computer s COM option UVS 99 under the system property hardware device manager is normal or not e Pull out the USB cable from the computer s USB port and re plug in e Install UVS 99 in another computer to rule out a faulty USB hub port on the original computer and then run the software If same thing happens please contact your local distributors 6 2 Optics System Checking If the pop up message Optic signal error appears when the UVS 99 is self checking the error could be due to the following reasons 37 e The life time of the Xenon flash lamp has been exhausted e The fiber is broken e The sample arm is open It must be closed e The position of source fiber and receiving fiber is not aligned Please contact your local distributors 6 2 Sample accurac
8. 00 310 320 330 340 350 360 Figure5 4 5 4 Light intensity adjustment The coefficient should be adjusted to get a continuous smooth intensity spectrum and keep the light intensity within 100 3500 It is also important to keep the intensity spectrum close to the reference spectrum which indicates the light source works well Figure 5 7 shows 7 intensity spectra with the coefficient from 1 to 7 Set the coefficient at 4 is more appropriate in this case since the spectrum is close to the reference spectrum and slightly lower 30 Settings 5 g imm 0 2mm coefficient pe 4000 Protein Bradford 3000 Cell Cultures j UV VIS j 2000 1000 0 Eg 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 Figure 5 5 It will lead to a jagged absorption spectrum if the light intensity is too high to get a continuous smooth curve On the contrary low light intensity will affect the absorbance measurement with high concentration samples 5 5 Special application A special measurement can be done by adjusting the coefficient of light intensity Here is an example of UV VIS measurement Both reference spectrum and intensityspectrum coefficient 1 of UV VIS module are shown below Figure 5 6 31 EEN EE SZ Settings B i Nucleic Acid Protein A280 _ Protein Lowry Protein Bradford 3000 Cell Cultures j 4000 2000 1000 200 Figure 5 6 From f
9. 1 2 5ul sample onto the measurement platform while the sampling arm is opened Lay down the sampling arm and make sure the solution bridges the gap between both optical fibers Click the button Measure and the measurement result will appear on the screen within 10 seconds Autosave The system will record and autosave the measurements The maximum number of records is 1000 When the measurement is completed open the sampling arm and wipe the sample from both pedestals using a laboratory wipe 8 Repeat the measurement till all samples are measured Click the button Report and all data would be exported into a new Excel table e Quick buttons There are two buttons on UVS 99 MEA is same as the Measurement icon in the operation software BLA is same as the Blank icon in the operation software Button Disable Button Enable Figure 3 2 3 3 Factors of affecting the measurement results e Samples carryover Any sample residue retained on the measurement platform would cause a slight error in the measurement result Clean both pedestals with a lab wipe right after the measurement is usually sufficient to prevent sample carryover Although generally not necessary 2ul water aliquots can be used to clean bothpedestals after particularly high concentration samples After measuring a large number of samples however it is recommended that the areas around the upper and l
10. 260 Sample absorbance at 260 nm represented as if measured with a conventional 10 mm path A260 280 Ratio of sample absorbance at 260 and 280 nm Concentration mg ml Concentration 1Abs 1 mg ml A280x 10 Concentration BSA A280x 10 6 7 Concentration IgG A280 x 10 13 7 Concentration Lysozyme A280 x 10 26 4 15 Autosave The system will record and autosave the measurements The maximum number of records is 1000 Save Graph Click Save Graph to save your graph if necessary Report Click Report and all results have been recorded will be exported to an Excel file Absorbance data shown in files are represented as displayed on the software screen A B E D E F G 1 Sample ID Abs260 Abs280 Abs280 260 280 concentratioi Sample Type 2 1 38 083 84 677 84 677 0 44 34 7 1 Abs 1mg ml 3 2 38 907 85 501 85 501 0 46 85 5 1 Abs lmg ml 4 3 40 629 87 223 87 223 0 47 87 2 1 Abs 1mg ml 5 4 42 351 88 945 88 945 0 48 88 9 1 Abs Img ml 6 5 44 073 90 667 90 667 0 49 90 7 1 Abs 1mg ml 6 45 795 92 389 92 389 0 50 92 4 1 Abs 1mg ml 8 7 46619 93 213 93 213 0 50 93 2 1 Abs 1mg ml Figure 4 4 16 4 3 Cell Cultures The Cell Cultures module displays the sample spectrum from 200nm to 850nm Two cursors can be set to the wavelengths of interest e Sample Volume Requirements 1 2 5 pl minimum 2 ul is recommended e Measurement UVS 99 will measure each s
11. 9 22 033 1 33 2 04 1484 ssDNA 15 b2 50 846 31 667 20 667 1 61 2 46 1677 9 ssDNA 16 b3 47 284 25 611 17 838 1 85 2 65 1560 4 ssDNA _17 b4 54 752 28 888 19 349 1 3 2 83 1806 8 ssDNA 18 b5 45 878 31 363 21 064 1 46 2 18 1514 ssDNA 19 b6 53 785 35 534 23 967 1 51 2 24 1774 9 ssDNA 20 b7 44 195 29 284 19 808 1 51 2 23 1458 4 ssDNA 21 b8 56 161 38 376 24 573 1 46 2 29 1853 3 ssDNA 22 b9 51 853 31 251 19 795 1 66 2 62 1711 1 ssDNA 23 b10 56 785 31 563 21 24 1 8 2 67 1873 9 ssDNA 24 Jbll1 44 788 32 227 24 194 1 39 1 85 1478 ssDNA 25 b12 47 898 32 563 22 448 1 47 2 13 1580 6 ssDNA 26 cl 28 013 15 947 10 964 1 16 2 55 924 4 ssDNA EE 18 932 14 468 9 229 1 31 2 05 624 7 ssDNA 28 c3 25 919 20 823 13 71 1 24 1 89 855 3 ssDNA 29 c4 33 563 22 053 14 046 1 52 4 39 1107 6 ssDNA Figure 4 2 4 2 Protein A280 e Sample Volume Reduirements 1 2 5 ul a minimum of 2ul is recommended e Range 0 1 75 Abs e Reproducibility 0 1 5 Abs 0 1 13 5 75 Abs 2 e Measurement UVS 99 will measure each sample with bothimm and 0 2mm path lengths and then automatically pick the appropriate one to normalize to a 10 mm path Protein A280 Absorbance 10mm 10 0 Blank i 30 80 Measure i I Save Graph 7 0 60 Report Record Exit P Button Disable 220 230 240 250 260 270 280 20 300 310 30 330 MY 350 360 Wavelength nm Gvans same C 9 fzo noe
12. MITATION DIRECT INDIRECT INCIDENTAL SPECIAL OR CONSEQUENTIAL DAMAGES INCLUDING BUT NOT LIMITED TO LOSS OF PROFITS REVENUE OR BUSINESS RESULTING FROM OR IN ANY WAY RELATED TO THE PRODUCTS These limitations apply regardless of whether such damages are sought based on breach of contract negligence strict liability in tort or any other legal theory The liability of The Manufacturer relating to any order by Customer shall in no event exceed the total amount of the payments received by The Manufacturer relating to such order By placing their order Customers represent and warrant that they and their employees and agents shall comply with all applicable laws ordinances regulations and codes in handling storage and use of Products and shall indemnify and hold harmless The Manufacturer from any claims rights or causes of action related thereto or to Customer s failure act in accord with these terms and warranties While information and data presented are accurate and reliable to the best of The Manufacturer s information and belief The Manufacturer does not guarantee the accuracy of any information and data nor do we represent that 43 reliance upon such information and data will ensure compliance with any applicable laws and regulations The Manufacturer reserves the right to alter these terms and conditions at any time and without notice to Customer Any variance of these terms is not valid unless it
13. ample with both1 mm and 0 2 mm path lengths and then automatically pick the appropriate one to normalize toa 1 mm ical Cell Cultures absorbance 10 0 gan Save Graph Report Record Ex Button Disable 200 250 300 350 400 Wavelength nm vans Max Absorbance _ 10 Wavelength1 350 Abs o UVS 99 Sample ID 9 Wavelength2 soo Abs dj Figure 4 5 Type the Sample ID Sample ID Used to define a name of sample 417 M Abs User selected wavelength and corresponding absorbance The wavelength can be selected by typing in or using the up down arrows to the left of the wavelength box 2 Abs User selected wavelength and corresponding absorbance The wavelength can be selected by typing in or using the up down arrows to the left of the wavelength box Max Absorbance Used to re scale the upper limit of the Y axis Autosave The system will record and autosave the measurements The maximum number of records is 1000 Save Graph Click Save Graph to save your graph if necessary Report Click Report and all results have been recorded will be exported to an Excel file Absorbance data shown in files are represented as displayed on the software screen 18 4 4 UV VIS The UV VIS module allows the UVS 99 spectrophotometer to function as a conventional spectrophotometer Sample absorbance is displayed on the screen from 200
14. art the UVS 99 software after saving the setting There are six measurement modules in the software Each module contains two coefficients for 0 2 mm and 1 mm You should adjust and save all the twelve coefficients respectively 28 5 3 Light intensity check The reproducibility of light intensity can be checked according to the spectra Please click the Read Date button continuously under the same coefficient to get severalspectra If all intensity spectra nearly coincide it indicates that the instrument works well with high reproducibility Figure 5 3shows 10 spectra black under the same coefficient and all intensity spectracoincide well Settings x Protein A280 j Protein Lowry Mimm 02mm coefficient sl pm 4000 Protein Bradford Cell Cultures UV VIS 2000 1000 cmg 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 Figure5 3 If the spectracannot coincide well Figure5 4 there are some problems in the photo source or optical fibers You can scour both pedestals with de ionized water to solve this problem If the spectra cannot coincide well after cleaning please contact your local distributor 29 X imm 0 2mm coefficient Road Data Settings Protein A280 Protein Lowry 4000 Protein Bradford 14 Cell Cultures UV VIS 2000 1000 e 220 230 240 250 260 270 280 290 3
15. b c Where A is the absorbance represented in absorbance units A E is the wavelength dependent molar absorptivity coefficient or extinction coefficient with units of liter mol cm b is the path length in cm and c is the concentration of sample in moles liter or molarity M To edit the calibration parameter of UVS 99 K is equivalent to editing the value b in the above formula so as to get more accurate A value Parameter Settings X 34 K1 is the calibration parameter of low absorbance value smaller than or equal to 10Abs 10mm It is recommended to use 4 8Abs 10mm standard sample or so for calibration e g double strand DNA 250ng ul K2 is the calibration parameter of high absorbance value bigger than 10Abs 10mm It is recommended to use 20 40Abs 10mm standard sample or so for calibration e g double strand DNA 1000ng ul When you click load icon you can find the original parameter for this UVS 99 Parameter Settings PS K1 0 9 Save toad M 35 For example when UVS 99 is used to measure certain standard sample and the real sample value is 100 however the measured value is 80 lower than 100 Then the user can change the calibration parameter to 100 80 1 25 Note Because the calibration parameters of each UVS 99 are all different please do take into account the original K parameter before editing the K parameter Taking the above case for example if
16. cluding dsDNA ssDNA RNA and other nm Abs User selected wavelength and corresponding absorbance The wavelength can be selected by typing in or using the up down arrows to the left of the wavelength box Note The user selected wavelength and absorbance are not utilized in any calculations A260 Absorbance of the sample at 260 nm represented as if measured with a conventional 10 mm path Note This is 10X the absorbance actually measured using the 1 mm path length and 50X the absorbance actually measured using the 0 2 mm path length A280 Sample absorbance at 280 nm represented as if measured with a conventional 10 mm path Note This is 10X the absorbance actually measured using the 1 mm path length and 50X the absorbance actually measured using the 0 2 mm path length 260 280 Ratio of sample absorbance at 260 and 280 nm The ratio of absorbance at 260 and 280 nm is used to assess the purity of DNA and RNA A ratio of 1 8 is generally accepted as pure for DNA a ratio of 2 0 is generally accepted as pure for RNA If the ratio is appreciably lower in either case it may indicate the presence of protein phenol or other contaminants that absorb strongly at or near 280 nm 260 230 Ratio of sample absorbance at 260 and 230 nm This is a secondary measure of nucleic acid purity The 260 230 values for pure nucleic acid are often higher than the respective 260 280 values They are commonly in the range of 1
17. d The sample intensity lsample along with the blank intensity Ibiank are used to calculate the sample absorbance Abs according to the following equation Abs og sem ee Thus the measured light intensity of both the sample and of the blank are reguired calculating the absorbance at a given wavelength 8 3 Concentration Calculation Beer Lambert Law The Beer Lambert equation is used to correlate the calculated absorbance with concentration A E b c Where A is the absorbance represented in absorbance units A E is the wavelength dependent molar absorptivity coefficient or extinction coefficient with units of liter mol cm b is the path length in cm and c is the concentration of sample in moles liter or molarity M 46 8 4 Fluorescent Dyes Measurement The UV VIS module of UVS 99 can be used to measure the absorbance of fluorescent dyes as well It can eliminate potentially flawed sample and improve research effectiveness in microarray application Based on the general form of the Beer Lambert equation you can calculate fluorescent dye concentrations with the measured absorbance The table of extinction coefficients for each dye is below Dye Type Extinction Coefficient Measurement liter mol cm Wavelength nm Cy3 150000 550 Cy5 250000 650 Alexa Fluor 488 71000 495 Alexa Fluor 546 104000 556 Alexa Fluor 555 150000 555 Alexa Fluor 594 73000 590 Al
18. d helps you install software for Ocean Optics USB2000 If your hardware came with an installation CD or floppy disk insert it now What do vou want the wizard to do Install the software automatically Recommended Install from a list or specific location Advanced Click Next to continue Figure 2 5 e Your UVS 99 should be ready for operation 2 3 Precautions 1 Before the UVS 99 software is installed in the computer do not turn on the power switch 1 is on and 0 is off And do not connect the USB cable to the computer 2 Do switch off the power 0 is off first before connecting or disconnecting the USB cable TO OFF the computer 3 Do not connect USB cable or do disconnect the USB cable to off the computer either when booting or shutting down the computer Do turn off the power and disconnect the USB cable off the computer when UVS 99 is not going to be used Do turn off the power and disconnect the USB cable off the computer before installing any kind of software in the computer The violation of all of the above precautions are deemed as misuse and not entitled to warranty The interval between each measurement must be more than 10 15 seconds If the sampling arm does not bounce back please switch off the power button immediately and re start the software 3 General Operation 3 1 Sample size recommendation eNucleic acid samples 1 2 5 ul eProte
19. e concentration value of the sample will be shown in Concentration in the module of standard 25 Protein Low Standard x gans Abs Concentration ma 7 1 Load Std f Save Std f Measure De ord ET Tr f Save Graph f Set Scate f Save Graph f Report i Concentration I Record 320 0 Exit P Button Disable Figure 4 14 Sample ID Used to define a name of sample wavelength 1 User selected wavelength and corresponding absorbance The wavelength can be selected by typing in or using the up down arrows to the left of the wavelength box The absorbance is related to the curve that selected to show wavelength 2 User selected wavelength and corresponding absorbance The wavelength can be selected by typing in or using the up down arrows to the left of the wavelength box The absorbance is related to the curve that selected to show Max Absorbance Used to re scale the upper limit of the Y axis Save Graph Click Save Graph to save your graph if necessary Report Click Report and a dialog box will pop up 26 5 Check and Adjustment of Light Intensity 5 1 Introduction If an un smooth or ragged absorption spectrum still appears after cleaning both pedestals you should check the light intensity The light intensity can be checked and adjusted by using the Settings module in the software There is no sample is needed when y
20. estals The metal sample pedestals are made from stainless steel and are resistant to most common laboratory solvents 7 6 Warranty Terms Products are supplied for research use only The Manufacturer warrants the Products are free from defects in material and manufacture and conform to the specifications at the time of shipment Subject to verification by The Manufacturer if any Product fails to conform to the specifications or any defect in material or manufacture appears 1 for Products which are scientific instruments within 13 months from the date of shipment and 2 for related Consumable Products modules and parts within 30 days from the date of shipment The Manufacturer s entire liability and Customer s exclusive remedy shall be at The Manufacturer s sole discretion the repair or replacement of the defective Products A4 7 7 Limitations to Warranty Unless a separate service plan is purchased all regularly scheduled maintenance and repairs during the Warranty term shall be the responsibility of Customer This warranty does not cover any damage to Products due to misuse improper site selection and maintenance or operation by Customer or any others which is contrary to recommended instructions and design parameters for the Products This warranty does not cover damage or defects resulting from repair or relocation of the Products by anyone other than an Avans representative After the initial installation of
21. exa Fluor 647 239000 650 Alexa Fluor 660 132000 663 Cy3 5 150000 581 Cy5 5 250000 675 47 Avans Biotechnology Corp www avansbio com
22. gher concentration than a general spectrophotometer can measure Figure 1 1 1 2 Operation Simply pipette 1 2 5 ul sample onto the measurement pedestal and then lay down the sampling arm to contact with the liquid sample The liquid sample will bridge the gap between both optical fibers The gap is controlled to both 1mm and 0 2 mm paths UVS 99 can automatically complete the whole measurement process within 10 seconds A pulsed xenon flash lamp provides the light source and a spectrometer utilizing a linear CCD array used to analyze the light after passing through the sample The instrument is controlled by special software installed in a computer Drip Measurement Figure 1 2 1 3 Applications e Undiluted nucleic acid 2 3700 ng ul e Purified protein A2go 5100 mg ml e Lowry amp Bradford e Cell density e General UV VIS spectrophotometry 2 Installation 2 1 Computer requirements e Operation system Windows7 amp above eOffice Excel 2007 2010 amp above e USB port e Screen 2 2 Software installation The UVS 99 software must be installed onto the computer before the USB cable is connected The UVS 99 software is in the CD rom provided Please pay attention here Please use Administrator ID to log in the Microsoft operation system The installation procedures are detailed as follow e Insert the UVS 99 CD rom into the CDROM unit Copy and save the software on the CD rom p
23. igure5 6 it seems the coefficient should no more than 1 or the intensity will be too high to get a continuous curve at 470 550nm However the sample K2Cr207 has an absorbance at 350nm and low light intensity lt 1000 there will limit the detection range of high concentration samples It s necessary to adjust the coefficient to increase the light intensity at 350 nm When the coefficient is adjusted to 4 the light intensity increases Figure 5 7 and the absorption spectrum is smoother at 350 nm Figure 5 8 Note Increase the coefficient may lead to saturation of intensity and affect the absorbance measurement at some wavelengths In this case the intensity is saturated Figure 5 7 and it leads to a jagged absorbance curve Figure 5 8 at430 550nm 32 N X Settings 1mm 02mm coefficient FT Nucleic Acid j 4000 Protein A280 Protein Lowry Protein Bradford 3000 Cell Cultures j 2000 1000 0 200 250 300 350 400 450 500 550 600 650 700 750 800 850 ar Mad Eg E Figure 5 7 U immKea 350 400 450 500 550 600 Figure 5 8 33 5 6 Set up of the Calibration Parameters Please open the CPSetting exe in the UVS 99 installation directory It can be used to modify and edit the absorbance calibration parameters and thus obtain more accurate measurement values Definition The Beer Lambert equation is used to correlate the calculated absorbance with concentration A E
24. in samples 1 2 5 ul e Suspended cell samples 1 2 5ul eOther samples 2 5 ul 3 2 General measurement protocol Check the connection between UVS 99 and the computer Turn on the power on off switch Make sure the sampling arm is closed and run the software UVS 99 will perform self check procedure Select a module from the main menu according to the sample type Figure 3 1 and UVS 99 will perform self check procedure again avans E Protein 280 4 R 7 Settings L Protein Bradford Figure 3 1 e Blanking Each time a software module is opened initiated the Measure button is inactive A blank must first be measured before the Measure button becomes active Lift the sampling arm then pipette 1 2 5ul of the blank sample the buffer solvent or carrier liquid used with your samples onto the measurement platform Lay down the sampling arm and make sure the solution bridge the gap between both optical fibers Click the button Blank UVS 99 will measure the solution with both1 mm and 0 2 mm path lengths and then the system will record both results automatically No need to re blank unless samples are in different solutions When the measurement is complete open the sampling arm and wipe the blanking buffer from both pedestals using a laboratory wipe e g Kimwipe e Sample measurement Type the Sample ID and select the Sample Type Pipette
25. is in writing and signed by an officer or other authorized representative of The Manufacturer The warranty is void if The Product has been abused or misused or if repairs have been attempted by unauthorised persons 44 8 Appendices 8 1 Instrument specifications Standard path length 1mm Short path length for high 0 2 mm concentration sample Sample volume 0 5 2 5 ul Light source Xenon flash lamp Detector 3648 element linear silicon CCD array detector Wavelength range 200 850 nm Wavelength accuracy 1 nm Wavelength resolution 3 nm FWHM at Hg 546 nm lt 1 8 nm FWHM at Hg 253 7 nm Absorbance precision 0 002 1 mm path Absorbance accuracy 2 at 0 76 absorbance at 257 nm Measurement accuracy 0 1 5 Abs 0 1 5 75 Abs 2 Measurement cycle time lt 5 seconds Size W20 D15 H12 cm Sample pedestal material Stainless steel Optical fiber cable material Quartz Voltage 12 VDC Power 6W Detection range of dsDNA 2 3500 ng ul Detects protein up to 100 mg ml Weight 1 4 kg 45 8 2 Absorbance calculation When the UVS 99 Spectrophotometer is blanked a spectrum is taken of a reference material blank and stored as an array of light intensity by wavelength When a measurement of a sample is taken the intensity of light that has transmitted through the sample is recorde
26. measurement wipe the sample from both the sampling arm and measurement pedestal simply using an ordinary dry laboratory wipe A final cleaning of both pedestals with de ionized water is also recommended after the user s last measurement 7 2 Parts that require replacements In general the only part that should periodically require replacement is the flash lamp The flash lamp theoretically can last a minimum of 300K measures There is no way to test the flash lamp to confirm its remaining life When the flash lamp fails the light output will become very erratic or stop altogether Please contact your local distributor if this happens 7 3 Calibration of wavelength The wavelength is auto calibrated based on known peaks in the xenon lamp spectra each time the software is started No calibration is required under normal use please contact your local distributor if needed 40 7 4 Solvent Compatibility The UVS 99 is compatible with most solvents typically used within sample solution in life science laboratories However all forms of Hydrofluoric Acid HF are incompatible as the fluoride ion will dissolve the quartz fiber optic cable 7 5 Decontamination of Measurement amp Optical Surfaces If decontamination is necessary a sanitizing solution such as a 5 25 solution of sodium hypochlorite freshly prepared bleach can be used to ensure that no biologically active material is present on the measurement ped
27. n arrows to the left of the wavelength box The absorbance is related to the curve that selected to show Max Absorbance Used to re scale the upper limit of the Y axis Save Graph Click Save Graph to save your graph if necessary Report Click Report and a dialog box will pop up 24 4 6 Protein Bradford The wavelength range for Bradford is between 575nm and 750nm 575nm is the major wavelength The default wavelength in the software is 595nm and 725nm The user can select his own wavelength e Sample Volume Requirements 1 2 5 ul minimum 2 ul is recommended e Measurement Taking the kit of Protein Bradford for example according to the kit protocol please use seven standards and set wavelength 1 as the major wavelength Measure and input the absorbance value of each sample by UVS 99 as shown in Figure 4 13 Then click standard and OK icon Protein LOWTY Absorbance 10 Record Sangle D AbWav abiwa Ban A Measure 2 3 4 5 6 on j Standard Save Graph Report Record Exit P Button Disable 0 45 450 475 500 5S 550 5 GO 625 650 675 70 75 750 Wavelengthinm Max Absorbancel 1 Wavelengthi z 650 Abs 001 Sample ID i Wavelength2 725 Abs Figure 4 13 Furthermore to the real samples please set wavelength 1 as the major wavelength The measured value from the major wavelength will be carried forward to the standard curve Th
28. ng large molecules such as genomic or lambda DNA are particularly susceptible to this phenomenon Note Larger volumes used by cuvette based spectrophotometers will minimize or mask the effect of sample non homogeneity e Confirm that reference blank solution and solvent are the same material Buffers often absorb in the UV range Therefore it is critical to blank the instrument with exactly the same material that the sample is suspended in e Confirm that your sample is not too dilute Measuring samples at or near the detection limit will result in measurements that can vary in a significant amount Refer to the applicable measurement range of the application module that you are using 6 3 Unusual spectrum A sample that exhibits jagged absorption spectrum but a normal shape may be the result of detector saturation This can be due to a dirty sample pedestal upon startup Try cleaning both sample pedestals thoroughly and restarting the software A spectrum that is very un smooth or ragged can also be caused by insufficient light intensity reaching the spectrometer If you suspects that this is occurring please refer to Section 5 Check and Adjustment of Light Intensity and adjust the intensity coefficient 39 7 Maintenance and Warranty 7 1 Cleaning To prevent sample carry over the most important thing is to keep the sampling arm and measurement pedestal clean Upon completing each sample
29. nm to 850nm and cursors permit the measurement of individual peaks e Sample Volume Requirements 1 2 5 ul minimum 2 ul is recommended e Measurement UV VIS Absorbance immed P 0 2mm Yellow 1 0 Blank 0 9 Measure 08 07 Standard 06 Report Save Graph 04 03 Record 02 Exit i A Button Disable 05 01 200 2 30 350 400 450 500 550 600 650 700 750 800 850 Wavelength nm av ans Max Absorbance 1 Wavelength1 350 Abs 584 UVS 99 Sample ID Wavelength2 soo Abs Figure 4 6 Type the Sample ID and select the Sample Type 19 Sample ID Used to define a name of sample Imm curve Show the curve of absorbencies measured by 1 mm path 0 2mm curve Show the curve of absorbencies measured by 0 2 mm path M Abs User selected wavelength and corresponding absorbance The wavelength can be selected by typing in or using the up down arrows to the left of the wavelength box The absorbance is related to the curve that selected to show 2 Abs User selected wavelength and corresponding absorbance The wavelength can be selected by typing in or using the up down arrows to the left of the wavelength box The absorbance is related to the curve that selected to show Note If both curves are shown the absorbance and the curve showed on the screen are related to 1 mm path Max Absorbance Used to re scale the upper limit of the Y axis
30. on values which simultaneously correspond to the X Y axis diagram in the left hand side After the absorbance and concentration value are keyed in click the icon of OK gt the Figure 4 9 is seen sad UV VIS Standard f Blank Abs Concentration _ 59 000 Fia toaasta Savest f Measure i 2 03 standard mo E an ere Save Graph _ f Set Scale f Report Save Graph Concentration f Record 3200 0 f Exit 2590 A Button Disable vans UVS 99 Figure 4 9 The user can proportionately dilute their samples of known concentration and then measure their individual absorbance The maximum rows for the user to key inis seven After the user finishes keying in the measured concentration and absorbance gt click OK the Figure 4 10 is seen Scale Setting X Scale X Con Scale Y Abs FOK Face Figure 4 10 gt 90 After the standard curve is set up and plotted when the user measures the same samples the user can key in an absorbance value in the field of Abs of the Sample frame in the right lower part in Figure 4 10 Then the concentration value will be automatically shown in the Concentration filed of the Sample frame according to the plotted standard curve 4 5 Protein Lowry Taking the kit of Protein Lowry for example according to the kit protocol please use seven standards and set wavelength 1 as the major wa
31. ou perform this procedure 5 2 Operation With the sampling arm down select the Settings module from the main menu The window will appear as the figure below Settings a e Nucleic Acid s Protein A280 _ Protein Lowry Protein Bradford 3000 Cell Cultures UVVIS 4000 2000 1000 0 Es 3 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 Figure 5 1 Select the type of sample you are working with For example the window will show as figure 5 1 when Nucleic Acid is clicked 7 Once the path length is selected 0 2 mm or 1 mm a red reference spectrum will appear 7 VS gt X j P 0 2mm coefficient O mn Oozm _ coomeient o eu Settings 4000 Protein Bradford a Cell Cultures UV VIS 2000 1000 Ca 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 Figure 5 2 Input a positive integer in the blank space 1 20 usually 4 is a good start and then click Read Data The software will draw up a black spectrum under the coefficient that you have input The light intensity should be kept within 100 3500 for better reliability and stability though the detection limit of intensity is 10 forUVS 99 The intensity spectrum should be close to the reference spectrum and slightly lower You can increase the coefficient to bring the intensity spectrum up Click the Save button can save the appropriate coefficient Rest
32. ower pedestals should be cleaned thoroughly e Samples homogeneity Samples in un homogeneous solution particularly when using small volumes would cause significant deviation in data generated by using all measurement technologies including spectrophotometer e Effect of evaporation Evaporation of the sample during the measurement would account for 1 2 increase in sample concentration Highly volatile solvents such as hexane would likely evaporate before the completion of measurement Less volatile solvents such as DMSO can be used successfully 10 4 Measurement 4 1 Nucleic Acids e Sample Volume Requirements 1 2 5ul 1 5 ul is recommended e Range 0 1 75Abs e Reproducibility 0 1 5 Abs 0 1 5 75 Abs 2 e Measurements UVS 99 will measure each sample with both1 mm and 0 2 mm path lengths and then automatically pick the appropriate one to normalize to a 10 mm path N ucleic Acid Absorbance 10mm 100 240 250 260 270 280 280 300 310 330 30 30 W Wavelength nm ava NS SampleiD 230 Abs 0 260Abs L Sample Type dsDNA 260 280 o 280Abs od UVS 99 2601230 j Ong gan Save Graph Report Record EER P Button Disable Figure 4 1 Type the Sample ID and select the Sample Type Sample ID Used to define the name of the sample 11 Sample Type Used to select the type of nucleic acid being measured in
33. rovided to the computer Double click the setup program and click OK to continue the installation i UVS 99 Setup Hd F Welcome to the UVS 99 installation program Setup cannot install system files or update shared files if they are in use Before proceeding we recommend that you close any applications you may be running Exit Setup Figure 2 1 e Change directory Please pay attention here Because of the security control of Microsoft operation system please install the software to non system drive that is D Drive or E Drive Click this button to install UVS 99 software to the specified destination directory Directory C Program FilestUVS 99 Change Directory Exit Setup Figure 2 2 3 UVS 99 Setup x Destination File C Program Files UVS 99 UVS 99 User Manual CN doc N O L LILI Qq Figure 2 3 e Click OK to finish the installation e After installation connect UVS 99 unit with the computer by the USB cable provided and then Found New Hardware Wizard should pop up on the computer screen as shown below Found New Hardware X Ocean Optics USB2000 Figure 2 4 e Run the wizard to search the driver Select Install the software automatically Recommended Click Next and the installation will be completed automatically Found New Hardware Wizard Welcome to the Found New Hardware Wizard This wizar
34. velength Measure and input the absorbance value of each sample by UVS 99 as shown in Figure 4 11 Then click standard and OK icon X Protein Lowry absorbance se 10 Record Same D Abava ame san 09 1 2 3 4 5 Exit 400 45 450 475 500 523 550 575 600 625 650 675 70 725 750 Wavelengthinm Max Absorbance 1 Wavelengthi 650 Abs Ru Sample ID i Wavelength2 725 Abs 001 Figure 4 11 Furthermore to the real samples please set wavelength 1 as the major wavelength The measured value from the major wavelength will be carried forward to the standard curve The concentration value of the sample will be shown in Concentration in the module of standard 23 Protein Lowt Standard x ina Abs Concentration unik sun TY tosd Sto f Save Std f Measure sanded Standard Save Graph Set Scale f Save Graph Report o I Concentration 20 Concentration f Record 220 0 er P Button Disable Figure 4 12 Sample ID Used to define a name of sample wavelength 1 User selected wavelength and corresponding absorbance The wavelength can be selected by typing in or using the up down arrows to the left of the wavelength box The absorbance is related to the curve that selected to show wavelength 2 User selected wavelength and corresponding absorbance The wavelength can be selected by typing in or using the up dow
35. y and reproducibility If the results are seemingly inaccurate or not reproducible the reasons could be sample or aliquot non homogeneity or liquid column breakage It may be helpful to try the following to ensure representative results e Make sure the sampling surfaces are clean before starting the software module A dirty sample pedestal can cause erroneous absorbance even negative values and signal saturation It is always a good practice to clean the surfaces with de ionized water to remove any residue e The liquid column breakage can cause an abnormity result The sufficient samples 1 2 5ul can insure the liquid column is intact Generally the surface tension of protein solution is lower than other solution so the liquid column cannot form easily To troubleshoot pipette some de ionized water onto the bottom pedestal and lower the upper pedestal into the solution and let it sit for approximately 2 to 3 minutes Wipe with a clean lab wipe to dry both pedestals Fold a clean dry lab wipe over several times to increase its thickness Press the lab wipe firmly down on the lower pedestal and rub the pedestal with a folded lab wipe very aggressively at least 15 20 times 38 e Heat DNA samples to 55 C and vortex before measurement Due to the small volumes required by the UVS 99 it is extremely important to ensure that the sample being measured is homogeneous Field experience has shown that samples containi
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