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GeneQuant 1300 User Manual - GE Healthcare Life Sciences

1. he 04 06 OF LO Le L14 Lowry Calibration Standards 0 100 0 400 0 077 A 0 056 4 0 600 0 095 4 0 800 01324 0 10 1 000 1 400 01694 0 205 4 A he 04 06 08 LO Le 14 o a a n Version 1 1 Calibration Screen replicates off This shows the calibration values and allows standards to be measured or entered using the keypad numbers if calibration mode is manual Step 11 standards selected Insert the reference sample Press 0A 100 key This will be used for all subsequent samples until changed Step 12 standards selected Insert the standard use C to clear previously stored results before measuring Y mM E to measure the standard and store the result Repeat step 12 for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 standards manual selected When all standards are measured the OK box appears Press OK to accept the calibration and go to the Results screen see below OR Press Back to cancel selections and return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Step 11 standards selected Insert the reference sample Press 0A 100 key This will be used for all subsequent samp
2. Step 7 Dilution Factor known Enter a dilution factor by using the keypad numbers within the range 1 00 9999 OR Step 7 Calculate Dilution Factor Press the options key RIKMA Enter the volume of the sample range 0 01 9999 using the keypad numbers Press the down arrow Enter the volume of diluent range 0 01 9999 by using the keypad numbers Press OK to calculate the dilution factor and return to the Parameters screen or press Cancel Es to cancel selections Step 8 Select units of measurement using left and right arrows Options are ug ml ng ul ug l Press the down arrow Step 9 Enter the factor using the keypad numbers Range 0 001 to 9999 Press OK to enter the results screen or Cancel return to the Applications Folder Ese to Page 58 OOo 008 Absorbance Ratio Concentration 0 26 pa ml Parameters Print Graph Sample MHumber Save Method uto Print E Version 1 1 Results Screen Step 10 Insert the reference Press 0A 100 key This will be used for all subsequent samples until changed Step 11 Insert sample and press l Repeat step 11 for all samples The absorbance at selected wavelengths is measured and the ratio between wavelengths 1 and 2 is calculated both corrected by the background wavelength value if this was selected Press to return to the Applications Folder Press KHK to display available Options which are described
3. mi 0 058 A osea man 0 058 A D OB i 14 DE DB LO LE LY Bradford Calibration Standards 0 200 0 058 A 0 400 0 196 A 0 600 0 335 A l 0 800 0463A T 4 1 000 0 592 4 n 1 400 orisa oe HE 04 06 OF LO Le L4 o Version 1 1 Calibration Manual entry Shows previously entered calibration values and allows values to be entered via the keypad The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Pressing the down arrow from the last standard will bring up the OK box Press OK to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Results screen Step 14 Insert the reference sample and press the 0A 100 T key This will be used for all subsequent samples until changed Step 15 Insert the sample and press MW The concentration of the sample is taken and displayed Repeat step 15 for all samples Press Esc to return to the Protein Folder Press KHM to display available Options which are described below Options select using key pad numbers Return to parameters screen step 1 above 2 Print result via selected method 3 Toggle graph on off Displays the calibration graph cursors give values for last measur
4. to return to the Life Science folder Press EEx to display available Options which are described below Version 1 0 Page 15 00 000 Version 1 0 Parameterz Print Graph Sample MHumber Save Method Anuto Print Exit options by pressing Options select using key pad numbers Return to parameters screen step 1 above Print result via selected method Toggle graph on off The graph shows a wavescan plot across the range 220 nm to 320 nm with cursors denoting 230 260 280 and if background correction selected 320 nm Sample number add a prefix to the sample number and reset the incrementing number to the desired value Save method use the alpha numeric keys to enter a name for the method and press Save Auto print toggles auto print on off Ese or wait Page 16 3 Oligo The procedure is as follows Oligo Parameters Step 1 Pathlength Units Press 3 to select Oligo mode Step 2 Select path length using the left and right arrows Options are 5 Dilution Factor Factor or 10 mm Press the down arrow Step 3 dilution factor known Background Enter the dilution factor using the keypad numbers Range 1 00 IE A to 9999 Use the C button to backspace and clear the last digit entered OF Step 3 calculate dilution factor Press KHK to enter the dilution factor screen Enter the volume of the sample using the keypad numbers Range 0 01 to 9999 Press the do
5. 10 mm a egin lt Step 3 dilution factor known Dilution Factor Factor Enter the dilution factor using the keypad numbers Range 1 00 to 9999 Use the C button to backspace and clear the last digit entered Background OR CIA Step 3 calculate dilution factor Press XII to enter the dilution factor screen see second a eee parameter screen to the left Enter the volume of the sample using the keypad numbers Range 0 01 to 9999 Press the down arrow Enter the volume of the diluent using the keypad numbers Range 0 01 to 9999 Press OK to calculate the dilution factor and return to the Parameters screen OR Press Cancel to cancel the selections and return to the Dilution Factor l Parameters screen Step 4 Volume 0 Select whether the background correction at 320 nm is used or not with the left and right arrows Press the down arrow Diluent e Step 5 Select the units of measurement using the left and right arrows Options ug ml ng ul ug pl Press the down arrow Step 6 Enter the factor using the keypad numbers Default value is 50 range is 0 01 to 9999 Step 7 Press OK to enter the Results screen and begin taking measurements OR to return to the Life Science folder DAA Parameters DAA Parameters Results Screen Step 8 Insert the reference sample Press 0A 100 T Key This will be used for all subsequent samples until changed Step 9 Insert sample and press E This measures at the se
6. AND WARRANTY austera asia idos 74 Version 1 0 Page 3 ESSENTIAL SAFETY NOTES There are a number of warning labels and symbols on your instrument These are there to inform you where potential danger exists or particular caution is required Before commencing installation please take time to familiarise yourself with these symbols and their meaning AN Caution refer to accompanying documents Background colour yellow symbol and outline black Unpacking Positioning and Installation e Inspect the instrument for any signs of damage caused in transit If any damage is discovered inform your supplier immediately e Ensure your proposed installation site conforms to the environmental conditions for safe operation Indoor use only Temperature range 5 C to 35 C Note that if you use the instrument in a room subjected to extremes of temperature change during the day it may be necessary to recalibrate by switching off and then on again once thermal equilibrium has been established 2 3 hours A temperature of no more than 4 C hour is recommended Maximum relative humidity of 80 up to 31 C decreasing linearly to 50 at 40 C e The instrument must be placed on a stable level bench or table that can take its weight lt 4 5 kg so that air can circulate freely around the instrument e This equipment must be connected to the power supply with the power adaptor supplied The adaptor can be used on 90 240 V 50 60 Hz supplies e I
7. Enter the day using the keypad numbers or left and right arrows Press the down arrow Day Hour Enter the month as above Enter the year Month Minute Press the down arrow Press the down arrow Year Enter the minute Seconds are zeroed when OK is pressed Press OK to store the settings and return to the Utilities folder es OR Press Cancel to return to the Utilities folder without storing the time Date and Time 2 Regional Sets Language and Number Format The procedure is as follows Regional Select a language Options are English French Spanish Italian Language or Japanese Press the down arrow Set the decimal point style Options are or 6 3 Humber Format Press OK to store the settings and return to the Utilities folder OR Press Cancel to return to the Utilities folder without storing the settings 9399 93 3 Printer Sets up printing options The procedure is as follows Select whether auto print is on or off using the left and right arrows When auto print is on the results are automatically Auto Print printed after a measurement is taken When it is off printing has to be initiated manually This can also be set using the Options key KHK in each application or method The default is OFF Printer Press the down arrow Select how the data are sent Options are Built in internal printer or to a computer via USB port or Bluetooth Printer Press OK to store the
8. History parameter is turned off all parameters and options will return to their default settings when you leave that application Unless it has been saved as a method Version 1 1 Page 42 1 Single Wavelength Abs and T This makes simple absorbance A and transmission T measurements on samples measuring the amount of light that has passed through a sample relative to a reference this can be air The procedure is as follows Single Wavelength Parameters Step 1 san Set wavelength by using keypad numbers or left and right arrows Range 190 1100 nm Press the down arrow key Mode Step 2 Select the mode Absorbance or T using the left and right arrows Step 3 To enter the results screen with the selected parameters press OK i OR Cancel the selections and return to the Applications Folder by pressing Cancel E Step 4 Insert the reference Press 0A 100 key This will be used for all subsequent samples until changed Step 5 Insert sample and press 1 Repeat step 5 for all samples Results The result at the selected wavelength is displayed on screen Use the left and right arrows to move the cursor and display the value at the cursor position 15nm from set wavelength Press Cancel to return to the Applications Folder Press KKM to display available Options which are described below Version 1 1 Page 43 Options select using key pad numbers l Parameters 0 Fr
9. Use the left and right arrows to select a folder to store the game in Favourites Methods 1 9 press the down arrow and enter name a S Press Cancel to return to the Utilities folder Version 1 0 Page 65 ACCESSORIES INSTALLATION Printer installation Version 1 0 Page 66 1 REMOVE THE POWER CABLE FROM THE INSTRUMENT Turn the instrument over and remove cap head screws from positions A and B using the Allen key provided 2 Turn the instrument back over and lift the accessory cover vertically upwards to remove Remove the tie wrap from the cable 3 Plug the accessory cable into the printer 4 Lower the printer onto the locating bosses and push down firmly 5 Invert the instrument and replace the cap head screws at A and B 6 Replace the top cover plate invert the instrument and replace the cap head screws at locations A and B shown in step 1 Printer 7 Switch the instrument on and go to utilities instrument preferences and select the Auto Print Built in printer Printer Version 1 0 Page 67 Loading changing the printer paper Lift off the paper cover Lock the platen and turn the knob to feed the paper Feed in the paper Sometimes it helps if the platen lock is released 5 Paper gripped 6 Cover replaced Version 1 0 Page 68 Bluetooth accessory installation 1 REMOVE THE POWER CABLE FROM THE INSTRUMENT Turn the instrument over and remove the cap head screws
10. absorbance measurements on the same sample The procedure is as follows Multi wavelength Parameters Step 1 Select the number of wavelengths Range 2 5 TERE Press the down arrow Step 2 Enter the first wavelength using either the number keys or the ail left and right arrows Press the down arrow Enter the second wavelength as above and repeat for the a2 number of wavelengths selected up to 5 Step 3 Press OK to enter the results screen OR Press Cancel to return to the Applications Folder Multi wavelength Step 4 Insert the reference Press 0A 100 key This will be used for all subsequent samples until changed Step 5 Insert sample and press a Repeat step 5 for all samples Multi wavelength Results A scan plot covering the range of wavelengths selected with cursors at the relevant wavelengths and a table of values is displayed alt ERI it A O I le O 300 400 500 Press to return to the Applications Folder A Press KHK to display available Options which are described halaw Options select using key pad numbers 1 Return to parameters screen step 1 above 2 Print result via selected method oO PIES 4 Print graph using selected method Grayed out if no data are GQ ri available 7 Sample number add a prefix to the sample number and GP Print Graph reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folde
11. below Options select using key pad numbers 1 Return to parameters screen step 1 above 2 Print result via selected method 3 Toggle graph on off Graph shows a wavescan plot across the selected wavelengths in place of the individual wavelength 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in Favourites Methods 1 9 press the down arrow and enter name 9 Auto print toggles auto print on off Exit options by pressing or wait Page 59 FAVOURITES AND METHODS FOLDERS These folders are the storage locations for any user modified Applications Methods that are saved in the Options menu Both are accessible from the home folders page Favourites This folder enables the user to quickly select any frequently used Methods Up to 9 Methods may be stored in the folder of eG ea Methods 1 Methods 2 Methods 3 of 9G ea Methods 4 Methods E Methods E of eG ob Methods Y Methods E Methods 3 Methods These are further storage folders enclosed in the top level Methods folder Up to 9 Methods may be stored in each folder Operation is identical to the Favourites Folder Saved methods can be locked unlocked and deleted using the Options menu Select the method by pressing the relevant key pad number and then press the HRM key Delete Method Press 1 to select d
12. data Set the t position starting point for the slope and dA calculation at the current cursor position Value is retained for subsequent samples Set the t position finishing point for the slope and dA calculation at the current cursor position Value is retained for subsequent samples Toggle the calculated slope line on and off Note if any data points enclosed by t and t are beyond the range of the instrument gt 2 5A or lt 0 3A then this option is greyed out Sample number add a prefix to the sample number and reset the incrementing number to the desired value Save method use the left and right arrows to select a folder to store in Favourites Methods 1 9 press the down arrow and enter name Auto print toggles auto print on off Ese or wait Page 52 5 Standard Curve The construction of a multi point calibration curve from standards of known concentration to quantify unknown samples is a fundamental use of a spectrophotometer this instrument has the advantage of being able to store this curve as a method using up to 9 standards To include a zero concentration standard include this in the number of standards to be entered and enter 0 00 for concentration use a reagent blank when required to enter the zero standard The procedure is as follows Standard Curve Parameters Step 1 Wavelength Curve Fit Select the wavelength using the keypad numbers or left and right arrows Range 190 11
13. for cleaning This can be removed by undoing the appropriate screws on the bottom of the instrument Version 1 0 Page 73 SPECIFICATION AND WARRANTY Wavelength range Monochromator Wavelength calibration Spectral bandwidth Wavelength accuracy Wavelength reproducibility Light sources Detector Photometric range Photometric linearity Photometric reproducibility Stray light Zero Stability Noise Digital output Dimensions Weight Power input 190 1100 nm Flat grating Automatic upon switch on 5nm 2 nm 1 nm Pulsed xenon lamp 1024 element CCD array 0 300 to 2 5004 0 to 199 T 0 005 Abs or 1 of the reading whichever is the greater 546 nm 0 003 Abs 0 to 0 5 Abs 0 007 Abs 0 5 1 0 Abs lt 0 5 at 220 nm and 340 nm using NaNO 0 01 Abs hour after 20 min warm up 340 nm 0 005 pk to pk 0 002 pms USB port standard Bluetooth option 260 x 390 x 100 mm lt 4 5 kg 18Vdc from a 90 250 V 50 60 Hz Max 30 VA mains power pack Specifications are measured after the instrument has warmed up at a constant ambient temperature and are typical of a production unit As part of our policy of continuous development we reserve the right to alter specifications without notice Warranty e General Electric Healthcare guarantees that the product supplied has been thoroughly tested to ensure that it meets its published specification The warranty included in the conditions of supply is valid for 12 months o
14. quantitating the binding of a dye Coomassie Brilliant Blue to an unknown protein and comparing this binding to that of different known concentrations of a standard protein at 595 nm this is usually BSA bovine serum albumin The Biuret method depends on reaction between Cupric ions and peptide bonds in an alkali solution resulting in the formation of a complex absorbing at 546 nm The BCA method also depends on reaction between cupric ions and peptide bonds but in addition combines this reaction with the detection of cuprous ions using bicinchoninic acid BCA giving an absorbance maximum at 562 nm The BCA process is less sensitive to the presence of detergents used to break down cell walls The Lowry method depends on quantifying the colour obtained from the reaction of Folin Ciocalteu phenol reagent with the tylsryl residues of an unknown protein and comparing with those derived from a standard curve of a standard protein at 750 nm this is usually BSA bovine serum albumin Detailed protocols are supplied with these assay kits and must be closely followed to ensure accurate results are obtained The use of plastic disposable cells is recommended To use a zero concentration standard include it in the number of standards to be entered and enter 0 00 for concentration use this when required to enter standard 1 A linear regression analysis of the calibration standard data points is calculated the result together with the correlation coeffici
15. retained for subsequent measurements To zoom out again use the down arrow Press to return to the Applications Folder Press KHK to display available Options which are described next Page 47 O a G O O o Peak Detection Auto detect Peaks Peak Detect on Zoom Hin Pk Height Sort Peaks By Min Pk width Draw Peaks Parameters Print Abs XT Peak Detection Add Peak Graph Scale Sample Humber Save Method A uto Pririt MW avescan 0 5 0 4 03 Version 1 1 A 496nm Abs 0 020 A Options select using key pad numbers 1 Return to parameters screen step 1 above 2 Print result via selected method 3 Toggle between Absorbance and T mode 4 Displays Peak Detection Parameter Screen See description below 5 Manually adds a peak position to the peak table in the results screen at the position set by the cursor If the cursor is returned to this position the legend User Defined Peak is displayed at the top of the scan and this option changes to Delete Peak 6 Displays Graph Scale Parameter Screen See description below 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the left and right arrows to select a folder to store in Favourites Methods 1 9 press the down arrow and enter name 9 Auto print toggles auto print on off Eso Exit options by pressing O
16. the keypad The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes 1 000 0 447 4 1 400 0 542 4 eg Press OK to accept the calibration and go to the Results Me MAU IE ME LI LE I screen see below OR Press Back to return to the Standards screen Bd Results screen Step 14 wavelength Insert the reference sample and press the 0A 100 T key This 548 nm will be used for all subsequent samples until changed Step 15 Absorbance ei Insert the sample and aros E The concentration of the sample is taken and displayed Repeat step 15 for all samples Curve Fit Regression Press to return to the Protein Folder Press KKM to display available Options which are described below Options select using key pad numbers 1 Return to parameters screen step 1 above E Parameters 2 Print result via selected method E Frin 3 Toggle graph on off Displays the calibration graph cursors P Graph give values for last measured sample 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the alpha numeric keys to enter a name Ed Sample Number for the method and press Save a aa 9 Auto print toggles auto print on off Auto P
17. the keypad numbers Range 0 001 to oe x Cancel e 9999 Use the C button to delete the last digit entered A A Press the down arrow key Step 3 if Standard is selected Enter the concentration using keypad numbers Range 0 01 9999 Use the C button to delete the last digit entered Press the down arrow key Step 4 Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Options key Kk and then use the left right arrows ug ml ug ul pmol ul mg dl mmol l mol l g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed decimal points DP to be selected from 0 to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 Concentration Parameters decimal point selected Press OK to store the chosen parameters or Cancel B Step 5 To enter the results screen with the selected parameters press Cancel the selections and return to the Applications Folder by pressing Cancel Eso Step 6 if using a Factor Insert the reference Press 0A 100 key This will be used for all subsequent samples until changed Step 7 Insert sample and press A The concentration of the sample is displayed Results shown as indicate the concentration is out of range Repeat step 7 f
18. ug ml in the case of RNA Oligonucleotides have a corresponding factor of 33 ug ml although this does vary with base composition this can be calculated if the base sequence is known Concentration Abs260 Factor The instrument uses factors 50 40 and 33 as defaults for DNA RNA and oligonucleotides respectively and compensates for dilution and use of cells which do not have 10 mm pathlength dilution factor and cell pathlength can be entered Nucleic Acid Purity Checks Nucleic acids extracted from cells are accompanied by protein and extensive purification is required to separate the protein impurity The 260 280 ratio gives an indication of purity it is only an indication however and not a definitive assessment Pure DNA and RNA preparations have expected ratios of gt 1 8 and gt 2 0 respectively deviations from this indicate the presence of impurity in the sample but care must be taken in interpretation of results The 260 nm reading is taken near the top of a broad peak in the absorbance spectrum for nucleic acids whereas the 280 nm reading is taken on a steep slope i e small changes in wavelength cause large changes in absorbance Consequently small variations in wavelength at 280 nm will have a greater effect on the 260 280 ratio than variations will at 260 nm Thus different instruments of the same and different tyoes may give slightly different ratios due to variations in wavelength accuracy But each instrument will giv
19. value is 0 76 range is O to 9999 Press the down arrow Step 6 Enter the coefficient value at 280 nm using the keypad numbers see method described in introduction Default value is 1 55 range is 1 to 9999 Press the down arrow Step 7 Select the units of measurement using the left and right arrows Options ug ml ng ul and ug pl Step 8 Press OK to enter the Results screen OR Cancel E to return to the Protein folder Page 28 00 DoS Version 1 1 Protein UW Parameterz Print Graph Sample MHumber Save Method Subo Frint Results Screen Step 9 Insert the reference sample Press 0A 100 T Key This will be used for all subsequent samples until changed Step 10 Insert a sample and press This measures at both 260 and 280 nm wavelengths and displays the result Protein concentration is calculated corrected by background wavelength value if selected Repeat step 10 for all samples Press F to return to the Protein folder Press KHK to display available Options which are described below Options select using key pad numbers 1 Return to parameters screen step 1 above 2 Print result via selected method 3 Toggle graph on off The graph shows a wavescan plot across the range 220 nm to 330 nm with cursors denoting 230 260 280 and if background correction selected 320 nm 7 Sample number add a prefix to the sample number and reset the incrementing number to
20. 00 nm Press the down arrow emer Enter the number of standard concentration points to be used in the curve 1 9 ae Step 3 dt a Pa Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Options key Kk and then use the left right arrows ug ml ug ul pmol ul mg dl mmol l mol l g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed decimal points DP to be selected from 0 to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters or Cancel E Standard Curve Parameters Step 4 E Select the type of curve fit using the left and right arrows wavelength Curve Fit Options straight line regression a zero regression this forces the straight line through the origin interpolated or cubic spline Step 5 Standards Calibration Select the calibration mode either Standards measure prepared standards or Manual keypad data entry Press the down arrow Units Replicates Step 6 if standards has been selected in step 5 Select the number of standards to be measured and averaged at each standard concentration point Can be OFF 1 2 or 3 Step Press Next to enter the Standards scr
21. 600 oooocnnccccnncnncccconccnnccncnnnnncccnnnnnnnnncnnnnnnnnnrnnnnnnnnrrnnnnnncnrnnnanans 25 TAPFOLEIN DETERMINATION ives ii A added ticos 27 o eC eee ee 28 27 A nC ee aCe ee eee 30 SO id 33 ANO WI A Nos dad 36 A ah thea gan ae ca Pi alee hte Tas a NA e cleat altace Ata sc Vi a healed etn ddI ata ial 39 THE APPEICATIONS FOLDER iii a e o ta eel LS 42 1 Single Wavelength ADS and Vii ecccsetasiecteceedcpectassccieedecciabedendcenstanicctes eociataseadivesecancenectinant 43 2 CONCA IN o eased iss eee a A 45 A arent a a 47 ASSIM Ple INIMGUICS ion 50 5 olandard CUVE ama ell odo o 53 6 MUDO WaVelend Mica ia ia 57 T ADSOance Ral sisi an 58 FAVOURITES AND METHODS FOLDERS sieniin tonnasi idad 60 UTLEIE S FOLDER aare A a a a aa wuecdons 61 T AUS ING TIME siiin ora ca 62 PAN oe A A A T 62 A O F eaa a a a aa N E e aa aa N aa ar a 62 o A a aE a Na aa aE a a r a a Na 63 A A a E a mdccauconavenedesaccauuerans 63 6 Folder NaMe S iria aaa 63 TADOU APA q e OC OO EENE NEE Eiaa 64 64 ACCESSORIES INSTALLATION cu arica ri weievedadsncanh eerduccwrueincs 66 PITA AMAN ds 66 Loading changing the printer a a 68 Bluetooth accessory SAA i 69 PRINT VIA COMP UTER arcon E a a a E 72 ACCESSORIE Susa as 73 After Sales SUDO dui 73 Lamp Replacement assiriana aaa a aaa aaa aa aAa a a a a a 73 Cleaning and general care of the INStrUMENL ccccesecesseeeeeeeeeeeeceesneeeeeeeeeeoensseeeeeseeooeenseeesseeooaeeenees 73 SPECIFICATION
22. GeneQuant 1300 USER MANUAL F viochrom Biochrom Ltd Certificate No 890333 Declaration of Conformity This is to certify that the GeneQuant 1300 UV Visible Spectrophotometers part numbers 80 2120 00 01 02 03 04 05 06 07 08 are manufactured by Biochrom Ltd conform to the requirements of the following Directives 73 23 EEC amp 89 336 EEC amp IVD Standards to which conformity is declared EN 61010 1 2001 Safety requirements for electrical equipment for measurement control and laboratory use EN 61326 2 3 1998 Electromagnetic compatibility generic emission standard Electrical equipment for measurement control and laboratory use EN 61000 4 6 1992 Electromagnetic compatibility generic immunity standard part 1 Residential commercial and light industry 2002 96 EC This appliance is marked according to the European directive 2002 96 EC on Waste Electrical and Electronic Equipment WEEE By ensuring this product is disposed of correctly you will help prevent potential negative consequences for the environment and human health which could otherwise be caused by inappropriate waste handling of this product The symbol R on the product or on the documents accompanying the product indicates that this appliance may not be treated as household waste Instead it shall be handed over to the applicable collection point for the recycling of electrical and electronic equipment Disposal must be carried out in accordan
23. MESS soo Applications Faw ourites Be Life Science Mi sthods LI tilities 4281 41 67119 Summary Function Keypad number Description el a 1 Nucleic acids Proteins and Cell counting Life Science N 2 Single wavelength Concentration Wavelength scan Kinetics Standard konein Curve Multiple wavelengths and Ratio e El ER 3 Sub folder for favourite user selected and configured methods Favourites oQ 4 Nine further sub folders for configured methods Methods EN 5 Instrument set up date time language etc and games Utilities Version 1 0 Page 8 Version 1 0 Page 9 LIFE SCIENCE FOLDER This contains seven applications subfolders Contents of these applicatons subfolders are detailed below of efi ora RNA afc of Tm Calculation Cy Ope oQ Protein Keypad number DNA Concentration and purity check for DNA samples RNA Concentration and purity check for RNA samples Oligo Concentration and purity check for Oligo samples TM Calculation DNA melting point calculator CyDye Labeling efficiency measurement OD 600 Cell culture OD600 with correction factor Protein Folder containing 5 methods of protein determination Version 1 0 Page 10 DNA RNA and oligonucleotide characterisation Nucleic Acid Quantification NAQ Nucleic acids can be quantified at 260 nm because it is well established that a solution of DNA in a 10 mm pathlength cell with an optical density of 1 0 has a concentration of 50 or 40
24. OS 5000 Parameters Print Graph Results if Sample Humber Save Method Anto Print Version 1 0 Results Screen Step 14 Insert the reference sample Press 0A 100 T Key This will be used for all subsequent samples until changed Step 15 Insert sample and press The system returns the dye concentration Repeat step 15 for all samples Press to return to the Life Science folder Press KRM to display available Options which are described below Options select using key pad numbers 1 Return to parameters screen step 1 above 2 Print result via selected method 3 Toggle graph on off The graph shows the spectral scan of the dye sample over the wavelength range of interest 4 Toggle results on off When off the results screen shows the absorbance values and absorbance ratios When on the results screen shows the total DNA DNA Yield nucleotides and number of dyes probe This only functions if the graph display is off see below 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the alpha numeric keys to enter a name for the method and press Save 9 Auto print toggles auto print on off Exit options by pressing F or wait Results screen with option 4 On Page 24 6 Bacterial Cell Culture Measurement OD600 e Bacterial cell cultures are routinely grown until the absorbance at 600 nm
25. a 04 06 OF 10 Le L4 0 4 A E HE 04 06 0E LO Le L4 Calibration Screen replicates off This shows the calibration values and allows standards to be measured or entered using the keypad numbers if calibration mode is manual Step 11 standards selected Insert the reference sample Press 0A 100 key This will be used for all subsequent samples until changed Step 12 standards selected Insert the standard use C to clear previously stored results before measuring ul dl to measure the standard and store the result Repeat step 12 for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 standards manual selected When all standards are measured the OK box appears Press OK HN to accept the calibration and go to the Results screen see below OR Press Back Esc to cancel selections and return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Step 11 standards selected Insert the reference sample Press 0A 100 key This will be used for all subsequent samples until changed Step 12 standards selected Press Replicates to display the replicate entry boxes Use C to clear previously stored results before measuring ie 7 to
26. a legend User T p A Defined Peak appears at the top of the graph The option then changes to Delete Peak to enable the user to remove the peak Note Storing a method at this stage will save these user defined wavelengths each time method is run Absorbance value at these wavelengths is reported Wf ave Scan User Defined Peak Cc fu Parameters Print Abs 327 Peak Detection Delete Peak Graph Scale Sample Muriber Save Method Auto Print O8000006 Graph Scale Shortcut button 6 lic This enables the user to set up a defined graph by defining the ad limits in either or both of the x and y axes Zoom mode This sets up the operation of the Zoom keys up and down arrows x amp y axes expands the display around the cursor measurement point whilst the other options select the absorbance or wavelength axes respectively With x or y axis limits set to on Zooming out will only be permitted to the set limits x y axis limits Setting x or y axis limits to On activates the start and finish points of the desired graph to user defined specific wavelengths and or absorbance values Pressing Cancel Eso ignores the selection pressing OK accepts them and displays the required graph Version 1 1 Page 49 4 Simple Kinetics Kinetics studies where the change in absorbance needs to be followed as a function of time at a fixed wavelength can be readi
27. a list of pre defined units press the Options key Kk and then use the left right arrows ug ml ug ul pmol ul mg dl mmol l umol l g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed decimal points DP to be selected from 0 to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Bradford Parameters Press OK to store the chosen parameters or Cancel Eso Step 5 Enter the type of curve fit Options are straight line regression zero regression forces the straight line through the origin interpolated or cubic spline Press the down arrow Bradford Parameters Step 6 METER Select the calibration mode either standards measure prepared standards or manual keypad data entry go to step 9 5395 nm 4 Regression Step 7 standards selected Sere ae Select the number of replicates using the left and right arrows This determines the number of standards to be measured and averaged at each standard concentration point Can be OFF 1 Units Replicates 2 or 3 SS i a as Press Next to enter the Standards screen OR Press Cancel to cancel selections and return to the Protein folder Standards Screen Step 9 standards manual selected Enter the concentration values by using the
28. and the use of reduced aperture cells The instrument can use background correction If it is used there will be different results from those when unused because Abs320 is subtracted from Abs260 and Abs280 prior to use in equations Concentration Abs 260 Abs 320 Factor Abs ratio Abs 260 Abs 320 Abs 280 Abs 320 Abs ratio Abs 260 Abs 320 Abs 230 Abs 320 If your laboratory has not used background correction before set this option to NO The use of background correction can remove variability due to handling effects of low volume disposable cells Version 1 0 Page 11 Spectral scan of nucleic acid Abs PURE NUCLEIC ACID POLY dAdT eters 260 0 Abril 700 1 a wa l L i Y 0 50 4 Fi Woo Waves240 0 Abra 303 4 f Se ks Fi ki h a A A A hy 2000 225 0 250 0 rs 300 0 325 0 350 0 Wear th Note e absorbance maximum near 260 nm and absorbance minimum near 230 nm e flat peak near 260 nm and steep slope at 280 nm e very little absorbance at 320 nm Operation of the instrument for Nucleic Acid measurements is described in the following sections DNA and RNA are very similar whilst in Oligo it is possible to calculate the factor from the composite bases by entering the proportions of the 4 bases Version 1 0 Page 12 1 DNA The procedure is as follows Step 1 Press 1 to select DNA mode Step 2 Select path length using the left and right arrows Options are 5 Pathlength Units or
29. ation of Christian and Warburg for the protein crystalline yeast enolase Biochemische Zeitung 310 384 1941 Protein mg ml 1 55 Abs 280 0 76 Abs 260 or Protein conc Factor 1 Abs 280 Factor 2 Abs 260 This equation can be applied to other proteins if the corresponding factors are known The instrument can determine protein concentration at 280 nm and uses the above equation as default the factors can be changed and the use of background correction at 320 nm is optional To customise the equation for a particular protein the absorbance values at 260 and 280 nm should be determined at known protein concentrations to generate simple simultaneous equations solving these provides the two coefficients In cases where Factor 2 is found to be negative it should be set to zero since it means there is no contribution to the protein concentration due to absorbance at 260 nm Set Factor 2 0 00 for direct A280 UV protein measurement Factor 1 is based on the extinction coefficient of the protein If BSA bovine serum albumin is an acceptable standard setting Factor 1 1 115 will give linear results from 0 to 0 8 mg ml protein Protein mg ml 1 115 Abs 280 Rapid measurements such as this at Abs 280 are particularly useful after isolation of proteins and peptides from mixtures using spin and HiTrap columns by centrifuge and gravity respectively Protein Determination at 595 546 562 and 750 nm The Bradford method depends on
30. ce with local environmental regulations for waste disposal RoHS Statement EU This device is currently exempt from the European RoHS requirements as it falls under category 9 of the Directive China This device does NOT fall under any categories detailed in the Chinese EIP classification list dated 16 3 2006 but for informational purposes currently is non compliant with Chinese RoHS requirements as it contains lead based solders but is safe for 20 years of use It is intended that lead free solder processes will be implemented as soon as possible Signed David Parr Managing Director Biochrom Ltd Biochrom Ltd 22 Cambridge Science Park Milton Road Cambridge CB4 OFJ England Telephone 44 0 1223 423723 Telefax 44 0 1223 420164 E mail enquiries biochrom co uk Website www biochrom co uk Registered in England No 3526954 Version 1 0 Page 2 TABLE OF CONTENTS ESSENTIAL SAFETY NOTES a o nos 4 Unpacking Positioning and Installation ori is 4 INTRODUCTION ovario ii iii odia 5 YOUF SHOCIODNO TO Melina A ii 5 Sample Nanding US nia A A 5 Keypad and diSDlaVy ii A A 6 TONE VE SU Oise eaters A o 8 LIFE SCIENCE FOLDER cuina a 10 DNA RNA and oligonucleotide characterisation c ccsscssccesssesseeeeeeesseeeeseoesseeeeseeesseeeesooeseeeeseonenseees 11 PE DIN ioecce cee otcuet accel dissed an 13 2 ANA oo 15 S DIO ia 17 ATi Calculadora a aaa 19 De CV ii A A A a a EAE 23 6 Bacterial Cell Culture Measurement OD
31. ck to cancel selections and return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Step 11 standards selected Insert the reference sample Press 0A 100 key This will be used for all subsequent samples until changed Step 12 standards selected Press replicates to display the replicate entry boxes Use C to clear previously stored results before measuring Insert the standard and press AM to measure the standard and store the result Repeat for all replicates and standards Press Next to move from replicates of one standard to replicates of the next standard A graph will display the results and the fitted curve as the measurements are input Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 standards manual selected Press OK to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Version 1 1 Page 34 Bradford Calibration Standards 0 700 0 059 A 0 400 0 156 4 0 600 0 331 A 0 300 1 000 1 400 8 p d 1 De LH 06 OB 20 Le L4 Bradford Calibration Standards 0 200 0 058 A 0 400 0 196 4 0 600 0 331 A 0 800 0463A 04 1 000 0 592 A 1 400 ora Pus me a n 04 06 08 10 Le L14 o Bradford Calibration Replicates 1 0 059 4
32. counter ion Na K TEA or Other Step 7 if Other selected Enter the molecular weight of the counter ion used Step 8 Press Next to select these parameters and go on to the Base Sequence screen OR Press Cancel to return to the Life Science folder Step 9 Select the pathlength of the sample cells 5 or 10 mm Press the down arrow Step 10 Enter the known base sequence triplets using the number keys 1 for A 3 for C 4 for G and 6 for T or U Step 11 Press OK to select these parameters and start to measure Tm OR Press Cancel Esc to return to the Parameters screen Page 21 SAO Results Screen Step 12 Insert the reference sample Press 0A 100 T Key This will be used for all subsequent samples until changed 0 750 A Step 13 115 1 Calculated Mw 44 o 1 S887 4 Insert sample and press E The unit measures the absorbance and uses this information to calculate the Measured Calculated Factor Tm 32 8 Repeat step 13 for all samples Press to return to the Life Science folder Press KRM to display available Options which are described below ERN Options select using key pad numbers E 1 Return to parameters screen step 1 above A 2 Print result via selected method 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the alpha numeric keys to enter a name Go Sample Number for the method and pre
33. creen Version 1 1 Page 54 Standard Curve Calibration Replicatez 00154 0 0154 0 10 ree TT CT ES Back Standard Curve Calibration Standards 10 0 0 0154 A 20 0 0 049 A 31 0 0 082 A D l2 10 62 0 01154 Holek amp Replicates 0 04 r 25 EE Back Standard Curve Calibration Screen replicates on This shows the calibration values and allows standards to be measured Step 10 Insert the reference Press 0A 100 key This will be used for all subsequent samples until changed Step 11 Press replicates to display the replicate entry boxes Use C to clear previously stored results before measuring Insert the standard and press H to measure the standard and store the result Repeat for all replicates Press Next to measure the next standard and repeat the process for all standards A graph will display the results and the fitted curve as the measurements are input Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 12 Press Next to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Calibration Manual entry Shows previously entered calibration values and allows values to be entered via the keypad The highlighted box can be edited in order to enter an absorbance value corresponding t
34. d is approx 10ul in a Quartz ultra micro cell e 12mm test tubes may be used e g for cell cultures however they are not recommended as higher quality data is produced by using disposable cuvettes for the analysis If used align the indicator line on 12 mm test tubes in the same direction to ensure reproducible positioning of the tube Note that test tubes do not last forever and that the surface becomes scratched and blemished through repetitive use if this is the case they should be replaced Version 1 0 Page 5 Keypad and display The back lit liquid crystal display is very easy to navigate around using the alphanumeric entry and navigation arrow keys on the hard wearing spill proof membrane keypad GeneQuant 1300 1 Confirm selection eQ Go to next screen o Life Science Applications Display screen o Methods eQ Utilities internal release only Arrow keys View options Key On off key Arrow keys View Options Bd BdM Alphanumeric keys Escape Set Reference 0A 100 T OK Next 1 Version 1 0 Action Turns the instrument on off Use the four arrow keys to navigate around the display and select the required setting from the active highlighted option View options for that application mode Some of these are common to all applications and described below Options unique to an application are described in the relevant section Use these to enter parameters and t
35. e Results screen see below OR Press Back F to return to the Standards screen Results screen Step 14 Insert the reference sample and press the 0A 100 T key This will be used for all subsequent samples until changed Step 15 Insert the sample and press i The concentration of the sample is taken and displayed Repeat step 15 for all samples Press F to return to the Protein Folder Press KRM to display available Options which are described below Options select using key pad numbers 1 Return to parameters screen step 1 above 2 Print result via selected method 3 Toggle graph on off Displays the calibration graph cursors give values for last measured sample 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the alpha numeric keys to enter a name for the method and press Save 9 Auto print toggles auto print on off Esc Exit options by pressing Or wait Page 32 3 Bradford The procedure is as follows Step 1 adela elite Press 3 to select Bradford method TT i The wavelength for this method is set at 595 nm Step 3 Enter the number of standard concentration points 1 9 to be used in the curve using the keypad numbers or left and right Standards Calibration P arrows Units E Press the down arrow a 2 Units The user can enter a text string up to 8 characters long To access
36. e consistent results within itself Concentration also affects 260 280 readings If a solution is too dilute the readings will be at the instrument s detection limit and results may vary as there is less distinction of the 260 peak and 280 slope from the background absorbance This is one reason why the Abs260 value should be greater than 0 1 for accurate measurements An elevated absorbance at 230 nm can indicate the presence of impurities as well 230 nm is near the absorbance maximum of peptide bonds and also indicates buffer contamination since This EDTA and other buffer salts absorb at this wavelength When measuring RNA samples the 260 230 ratio should be gt 2 0 a ratio lower than this is generally indicative of contamination with guanidinium thiocyanate a reagent commonly used in RNA purification and which absorbs over the 230 260 nm range A wavelength scan of the nucleic acid is particularly useful for RNA samples The instrument can display 260 280 and 260 230 ratios and compensates for dilution and use of cells that do not have 10 mm pathlength dilution factor and cell pathlength can be entered Use of Background Correction Background correction at a wavelength totally separate from the nucleic acid and protein peaks at 260 and 280 nm respectively is sometimes used to compensate for the effects of background absorbance The wavelength used is 320 nm and it can allow for the effects of turbidity high absorbance buffer solution
37. e reference sample and press the 0A 100 T key This will be used for all subsequent samples until changed Step 15 Insert the sample and press E i The concentration of the sample is taken and displayed Repeat step 15 for all samples Press to return to the Protein Folder Press KRM to display available Options which are described below Options select using key pad numbers O Parameters 1 Return to parameters screen step 1 above 8 Pi 2 Print result via selected method Graph 3 Toggle graph on off Displays the calibration graph cursors give values for last measured sample 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value E Sample Number 8 Save method use the alpha numeric keys to enter a name seve ene for the method and press Save El Auto Print 9 Auto print toggles auto print on off Eso Exit options by pressing Or wait Version 1 1 Page 38 5 Biuret The procedure is as follows Biuret Parameters Step 1 Press 5 to select Biuret method Curve Fit Step 2 The wavelength for this method is set at 546 nm Step 3 Enter the number of standard concentration points 1 9 to be el used in the curve using the keypad numbers or left and right arrows a Step 4 Units The user can enter a text string up to 8 characters long ACE amet To access a list of pre defined units press the Options key KAR and then us
38. e the left right arrows ug ml ug ul pmol ul mg dl mmol l umol l g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed decimal points DP to be selected from 0 to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters or Cancel Eso Step 5 Enter the type of curve fit Options are straight line regression zero regression forces the straight line through the origin A acid interpolated or cubic spline Press the down arrow EJE i 4 a 1 1 Select the calibration mode either standards measure prepared SEEE SEEEN standards or manual keypad data entry go to step 9 ekes Select the number of replicates using the left and right arrows TTE SEE This determines the number of standards to be measured and averaged at each standard concentration point Can be OFF 1 2 0r 3 Step 8 standards selected a Press Next to enter the Standards screen OR Biuret Standards Press Cancel to cancel selections and return to the Protein folder Standards Screen Step 9 standards manual selected Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range 0 001 to 9999 C button backspac
39. ect the calibration mode either standards measure Curve Fit prepared standards or manual keypad data entry go to step TT o Step 7 standards selected Calibration Select the number of replicates using the left and right Sa Se arrows This determines the number of standards to be measured and averaged at each standard concentration o a ha of Step 8 standards selected Next to enter the Standards screen Press Cancel Esc to cancel selections and return to the Protein folder CA Standards Standards Screen Step 9 standards manual selected Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range 0 001 to 9999 C button backspaces and clears the last digit entered Step 10 standards manual selected Std 3 Std 6 Press Next to enter the Calibration screen If there are duplicate or non monotonic increasing entries the unit will beep and highlight the incorrect entry OR Press Back to return to the Parameter screen Version 1 1 Page 30 BCA Calibration BCA Calibration Standards 0 200 0 050 4 Ala 04 06 08 140 Le L4 0 400 01m A 0 600 0 265 4 0 800 0 404 A 1 000 0 5764 1 400 0 626 A a L BCA Calibration Standards 0 200 0 050 4 0 400 a1r1 4 0 600 0 2885 0 200 0 404 4 1 000 0 5164 1 400 0 626 A Version 1 1 LJ m o l i 0 4 o ee a E na 04 06 OF 10 Le L4 i i
40. ed sample 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the alpha numeric keys to enter a name for the method and press Save 9 Auto print toggles auto print on off Eso Exit options by pressing Or wait Page 35 4 Lowry The procedure is as follows Lowry Parameters Step 1 ER Press 4 to select Lowry method ide The wavelength for this method is set at 750 nm Standards Calibration Step 3 f e Enter the number of standard concentration points 1 9 to be used in the curve using the keypad numbers or left and right Units Replicates arrows O Step 4 Units The user can enter a text string up to 8 characters long Lovery Parameters To access a list of pre defined units press the Options key Ex Ex and then use the left right arrows ug ml ug ul pmol ul mg dl mmol l umol l g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed decimal points DP to be selected from 0 to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters or Cancel Eso Step 5 Enter the type of curve fit Options are straight line regression zero regression forc
41. een OR Press Cancel to cancel selections and return to the Applications Folder Version 1 1 Page 53 Standard Curve Standards Standards screen Step 8 Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range 0 001 to 9999 Step 9 Press Next to enter the Calibration screen If any duplicate or non monotonic increasing entries are present the unit will beep and highlight the incorrect entry OR Press Back to return to the Parameter screen Standard Curve Calibration Calibration Screen replicates off This shows the calibration values and allows standards to be CC PE measured Step 10 Insert the reference Press 0A 100 key This will be used for all subsequent samples until changed Step 11 Insert the standard use C to clear previously stored results before measuring 1d m 30 340 SO E to measure the standard and store the result Repeat for all standards Standard Curve Calibration Spee Lu A graph will display the results and the fitted curve as the 10 0 aoma measurements are input 20 0 0 0073 A 0 05 Use the up and down arrows to select a standard to be repeated 0 007 A if a poor reading has been obtained Use C to clear the previous g reading Step 12 Press OK to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards s
42. elete method Select the method to be deleted using the left and right arrows Press to delete the method OR cancel to return to Sa E Lock Method o Press 2 to select lock method Se a Select the method to be locked using the left and right arrows Press the down arrow Select a pass code using the keypad numbers or left and right arrows P Delete Method Press to lock the method OR cancel F to return to the 8 Lock Method Favourites Methods folder E Unlock Method e Unlock Method Press 3 to select unlock method Select the method to be unlocked using the left and right arrows Press the down arrow Enter the pass code using the keypad numbers or left and right arrows Press to unlock the method OR cancel to return to the Favourites Methods folder Version 1 1 Page 60 UTILITIES FOLDER Press 5 to enter the utilities folder of ef ef Oate and Time Regional Printer eo o Contrast Folder Mames Summary Function Keypad number Description a 1 Set correct time and date Date and Time e 2 Select preferred language and number format Regional 3 Printer output options 4 Select screen layout themes and history Preferences 5 Adjust screen contrast amp brightness Contrast O al 6 Re name Method folders Folder Warnes i 7 Serial number and software version About o NT 8 Spectro Blocks Sudoku Version 1 0 Page 61 1 Date and Time The procedure is as follows
43. ent can be printed out A correlation coefficient of between 0 95 and 1 00 indicates a good straight line Version 1 1 Page 27 1 Protein UV This is the Christian and Warburg assay discussed above The procedure is as follows Protein UV Parameters Pathlength A260 Factor Dilution Factor 4280 Factor 1 000 1 550 Background Units a Protein UV Parameters Dilution Factor Yolume Diluent 0 000 Version 1 1 Step 1 Press 1 to select Protein UV mode Step 2 Select path length using the left and right arrows Options are 5 or 10 mm Press the down arrow Step 3 dilution factor known Enter the dilution factor using the keypad numbers Range 1 00 to 9999 Use the C button to backspace and clear the last digit entered OR Step 3 calculate dilution factor Press XII to enter the dilution factor screen shown to the left Enter the volume of the sample using the keypad numbers Range 0 01 to 9999 Press the down arrow Enter the volume of the diluent using the keypad numbers Range 0 01 to 9999 Press OK to calculate the dilution factor and return to the Parameters screen OR Press to cancel the selections and return to the Parameters screen Step 4 Select whether the background correction at 320 nm is used or not with the left and right arrows Press the down arrow Step 5 Enter the coefficient value at 260 nm using the keypad numbers see method described in introduction Default
44. ents The ACGT U sequence entered into the utility is used to calculate the theoretical T the theoretical absorbance Absorption units umol and the conversion factor ug ml This is because the stability of a bent and twisted sequence of bases such as an oligonucleotide is dependent on the actual base sequence the calculated thermodynamic interactions between adjacent base pairs have been shown to correlate well with experimental observations This utility uses matrices of known published thermodynamic values and extinction coefficients to calculate Tm and the theoretical absorbance factor respectively of an entered base sequence Tm This is calculated using the equation Te AH x 100 273 15 16 6 log salt AS 1 987 x log c 4 53 0822 where AH and AS are the enthalpy and entropy values summed from respective 2x4x4 nearest neighbour matrices c is the Primer concentration of oligonucleotide pmoles ul in the calculated Tm or the measured concentration in measured Tm In the latter case concentration is obtained from the equation ce Abs 260nm x calculated factor x pathlength multiplier x 10000 Calculated MW Calculated factor and MW are defined below salt is the buffer molarity plus total molarity of salts in the hybridization solution moles Weights for AS are indexed by adjacent paired bases A similar equation applies to Weights for AH again indexed by adjacent bases Note that bivalent salts may need normali
45. es and clears the last digit entered Step 10 standards manual selected Press Next to enter the Calibration screen OR Press Back to return to the Parameters screen Version 1 1 Page 39 Biuret Calibration ia E 04 06 OF LO Le L4 Biuret Calibration Standards 0 044 4 0 148 A 0 4 0 243 A 0 349 A 0 200 0 400 0 600 0 800 1 000 0447 a 0 542 4 a Li 1 400 Oot Es ld tr 2 04 06 08 LO Le L4 Biuret Calibration Fi o2 mi mE DE LO LE LY Biuret Calibration Standards 0 200 0 044 4 0 400 0 148 A 0 4 0 600 0 243 A 0 800 0 349 A 1 000 044r 4 ma 1 400 0 5424 Lu Es OL F E 02 DH 06 08 Lo Le Ly o Version 1 1 Calibration Screen replicates off This shows the calibration values and allows standards to be measured or entered using the keypad numbers if calibration mode is manual Step 11 standards selected Insert the reference sample Press 0A 100 key This will be used for all subsequent samples until changed Step 12 standards selected Insert the standard use C to clear previously stored results before measuring Y mM Press A E to measure the standard and store the result Repeat step 12 for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13
46. es the straight line through the origin oe one interpolated or cubic spline Press the down arrow Select the calibration mode either standards measure prepared Seas can standards or manual keypad data entry go to step 9 Step 7 standards selected Select the number of replicates using the left and right arrows E ENE This determines the number of standards to be measured and O at each standard concentration point Can be OFF 1 or 3 Step 8 standards selected in Press Next to enter the Standards screen OR Press Cancel to cancel selections and return to the Protein folder Lowry Standards Standards Screen Step 9 standards manual selected Enter the concentration values by using the keypad numbers and the up and down arrows to move between the different standard boxes Range 0 001 to 9999 C button backspaces and clears the last digit entered Step 10 standards manual selected Press Next to enter the Calibration screen If there are duplicate or non monotonic increasing entries the unit will beep and highlight the incorrect entry OR Press Back to return to the Parameters screen Version 1 1 Page 36 Lowry Calibration OOS ne O14 06 OF LO Le 14 Lowry Calibration Standards 0 100 0 400 0 077 A 0 056 A 0 600 0 054 A 0 800 01324 1 000 1 400 01694 0 205 4 0 05 A he 04 06 OF LO Le L14 Lowry Calibration Replicates 1 000174 000174 000174 0 017 A
47. f the instrument has just been unpacked or has been stored in a cold environment it should be allowed to come to thermal equilibrium for 2 3 hours in the laboratory before switching on This will prevent calibration failure as a result of internal condensation e Switch on the instrument via the keypad 10 after it has been plugged in The instrument will perform a series of self diagnostic checks e Please read through this user manual prior to use e Please contact your original supplier in the first instance if you experience technical or sample handling difficulties If this equipment is used in a manner not specified or in environmental conditions not appropriate for safe operation the protection provided by the equipment may be impaired and instrument warranty withdrawn Version 1 0 Page 4 INTRODUCTION Your spectrophotometer Your spectrophotometer is a simple to use UV Visible instrument with twin CCD array detectors 1024 pixels It has no moving parts which is the basis of the rapid scanning operating system The user interface is built around folders which are displayed on the home page when the instrument is switched on After switch on and calibration the default home page is GeneQuant 1300 offering the choice of Life Science Standard Life Science methods such as nucleic acid assays protein assays and cell counting Applications General spectroscopic methods Favourites A folder to store your more frequently
48. from positions A and B using the Allen key provided 2 Turn the instrument back over and lift the accessory cover vertically upwards to remove Remove the tie wrap from the cable 3 Plug the accessory cable into the Bluetooth module 5 Note the slots in the base of the case The two lugs on the Bluetooth module plug into these Version 1 0 Page 69 5 Load the rear cover into the two slots provided note the large flange is to face upwards Re fit the top plate 6 Invert the instrument and replace the cap head screws at A and B as shown in section 1 earlier 6 Attach the license label as shown Version 1 0 Page 70 6 Switch the instrument on and go to the preferences page under utilities instrument and select the Auto Print Bluetooth option Printer Printer 4 Computer Bluetooth F Version 1 0 Page 71 PRINT VIA COMPUTER e PVC Print Via Computer is a small application running under Windows 2000 Windows XP or Windows Vista to enable a GeneQuant 1300 to transfer data into a PC environment From there the user has a selection of choices the data can be both printed or saved in a variety of formats PVC is capable of supporting several instruments simultaneously limited only by hardware and the speed of the host system e PVC can operate via USB and Bluetooth simultaneously e PVC can store data either to a common directory or be configured to save to independent directories by bo
49. he down arrow Input the new name for the folder Folder Methods 1 Hew Hame Press OK to store the settings and return to the Utilities folder OR Press Cancel to return to the Utilities folder without storing the settings This folder allows you to rename the method or favourite folders Version 1 0 Page 63 7 About es Displays the instrument serial number and software version o D About GeneQuantT 1300 Press OK to close the window and return to the Utilities folder O Serial Number 54321 O Wersion 4281 1 6718 F o Games 8 Games Spectro Blacks Sudoku 1 Spectroblocks 1 Hew Game 2 Options 3 Exit Classic block dropping game Follow the instructions Press Cancel E to return to the Utilities folder without storing the settings Version 1 0 Page 64 2 Su Doku Can be set up as Computer mode 50 preset games or User mode enter your own pattern Use the cursors to select the square and the key pad to enter a number Invalid numbers cannot be entered Cells can be locked or unlocked by using the decimal point Unlocked cells can be cleared using the C key see also option key below The user mode starts with a blank grid Sudoku Setup Sudoku Game 1 Mode Computer Game Options Press KIM to display the options menu Return to the set up screen The instrument solves the game for you Clear all entries Save the game
50. he ratio of wavelengths 1 and 2 absorbencies are calculated both corrected by the background wavelength value if selected Gives concentration based on absorbance at wavelength 1 Repeat step 9 for all samples Press to return to the Life Science folder Press EEx to display available Options which are described below Options select using key pad numbers EP Parameters 1 Return to parameters screen step 1 above BO Pin 2 Print result via selected method Graph 3 Toggle graph on off The graph shows a wavescan plot across the range 220 nm to 320 nm with cursors denoting 230 260 280 and if background correction selected 320 nm E Sample Number 7 Sample number add a prefix to the sample number and Ed Save Method reset the incrementing number to the desired value Eh sute Print 8 Save method use the alpha numeric keys to enter a name for the method and press Save 9 Auto print toggles auto print on off Exit options by pressing or wait Version 1 0 Page 18 4 Tm Calculation This utility calculates the theoretical melting point from the base sequence of a primer It is done using nearest neighbour thermodynamic data for each base in the nucleotide chain in relation to its neighbour Breslauer et al Proc Natl Acad Sci USA Vol 83 p3746 1986 The data obtained are useful both in characterization of oligonucleotides and in calculating T for primers used in PCR experim
51. ient of extinction Press the down arrow Step 5 Enter the pathlength of the sample cell 5 or 10 mm Press the down arrow Step 6 Select whether background correction Is on or off Step 7 Press Next to enter the settings screen OR Press Cancel to return to the Life Sciences folder Step 8 dilution factor known Enter the dilution factor using the keypad numbers Range 1 00 to 9999 Use the C button to backspace and clear the last digit entered OR Step 8 calculate dilution factor Press XII to enter the dilution factor screen see second image to the left Enter the volume of the sample using the keypad numbers Range 0 01 to 9999 Press the down arrow Enter the volume of the diluent using the keypad numbers Range 0 01 to 9999 Press OK to calculate the dilution factor and return to the Parameters screen OR Press to cancel the selections and return to the Parameters screen Step 9 Enter the conversion factor using the keypad numbers Press the down arrow Step 10 Enter the volume of the probe in ul Press the down arrow Step 11 Enter the amount of starting RNA in ng Maximum 99999 ng Step 12 if background correction is on Enter the background correction wavelength if required Step 13 Press OK to select these settings and start to measure OR Press Cancel to cancel the settings and return to the parameters screen Page 23 AZGO AZ30 2 589 AZ60 AZ380 1 548 AdyefAZ60 1 046 D
52. in 1 Return to parameters screen step 1 above O 21 2 Print result via selected method GP Print Graph 3 Toggle between Absorbance and T mode 4 Print graph greyed out if no data are available 7 Sample number add a prefix to the sample number and MA Sample Number reset the incrementing number to the desired value Save Method 8 Save method use the left and right arrows to select a folder y A uto Print de D to store in Favourites Methods 1 9 press the down arrow and enter name 9 Auto print toggles auto print on off Eso Exit options by pressing Or wait Version 1 1 Page 44 2 Concentration This makes simple concentration measurements on samples by measuring the amount of light that has passed through a sample relative to a reference this can be air Concentration is obtained by multiplying the measured absorbance at a specific wavelength by a factor The factor may be known in advance or may be calculated by the instrument by measuring a standard of known concentration The procedure is as follows Concentration Parameters Step 1 Set wavelength by using keypad numbers or left and right Wavelength arrows Range 190 1100 nm Press the down arrow key Step 2 Mode Select the mode Factor user entered or Standard factor is Factor calculated from a calibration sample using the left and right arrows Factor Press the down arrow key Step 3 if Factor is selected Enter the Factor using
53. keypad numbers and the up and down arrows to move between the different standard boxes Range 0 001 to 9999 C button backspaces and clears the last digit entered Step 10 standards manual selected Bradford Standards Press Next to enter the Calibration screen If there are duplicate or non monotonic increasing entries the unit will beep and highlight the incorrect entry OR Press Back to return to the Parameters screen Version 1 1 Page 33 Bradford Calibration Calibration Screen replicates off E 5 This shows the calibration values and allows standards to be a measured or entered using the keypad numbers if calibration ar mode is manual ae E Step 11 standards selected a Insert the reference sample Press 0A 100 key This will be used for all subsequent samples until changed Step 12 standards selected Insert the standard use C to clear previously stored results Of 14 26 08 Lo L2 LY before measuring Press i to measure the standard and store the result Repeat step 12 for all standards A graph will display the results and the fitted curve as the measurements are made Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 standards manual selected When all standards are measured the OK box appears Press OK to accept the calibration and go to the Results screen see below OR Press Ba
54. known as OD600 reaches approximately 0 4 prior to induction or harvesting A linear relationship exists between cell number density and OD 600 up to approx 0 6 e ltis important to note that for turbid samples such as cell cultures the absorbance measured is due to light scattering and not the result of molecular absorption The amount of scatter is affected by the optics of the system distance between the cell holder and instrument exit slit geometry of this slit and the monochromator optics Different spectrophotometer types therefore give different responses for the same turbid sample to compare results they must be normalised using calibration curves e A calibration curve can be determined by comparing measured OD 600 to expected OD 600 Expected OD 600 is determined by counting cell number using an alternative technique for example microscope slide method and converting to OD 600 using the rule of thumb that 1 OD 600 8 x 10 cells ml for E Coli e Your GeneQuant instrument has much smaller optics than most conventional spectrophotometers and more light is transmitted through to the detector resulting in lower than expected OD 600 values Results obtained by comparing measured OD 600 with expected OD 600 see above indicate that a correction factor of 2 0 is required to make the data comparable to larger instruments this factor is included as a default value in set up e The use of 10 mm pathlength disposable cells is recommended f
55. lculated molecular weight a weight is added for each base looked up from a table The weight of the counter on is added for every base from a small table for the known ions If phosphorylated then the system adds 17 0 plus two counter ions otherwise it subtracts 61 0 and one ion Theoretical Absorbance For each adjacent pair of bases nearest neighbours a weight is accumulated using a table For each internal base a weight is subtracted using another table Separate tables are used for DNA and RNA Calculated factor This is just the calculated molecular weight divided by the theoretical absorbance Version 1 0 Page 20 Tm Calculation Parameters Base Type Buffer Molarity Phosphorylated Counter lon Primer Conc Tm Calculation Parameters Base Type Buffer Molarity Phosphorylated Counter lon Primer Conc Other Mw Tm Calculation Base Sequence Pathlength Base Sequence Tm Calculation Base Sequence Pathlength Base Sequence AGC AAG A CT Version 1 0 Step 1 Press 4 to enter the Tm Calculation parameters screen Step 2 Select the base type DNA or RNA Press the down arrow Step 3 Select whether the sample is phosphorylated or not yes or no Press the down arrow Step 4 Enter the primer concentration using the keypad numbers Range 0 000 to 99 9 Press the down arrow Step 5 Enter the buffer molarity using the keypad numbers Range 0 000 to 10 Press the down arrow Step 6 Select the
56. lected wavelengths and displays the results The ratio of wavelengths 1 and 2 absorbencies are calculated both corrected by the background wavelength value if selected Gives concentration based on absorbance at wavelength 1 Repeat step 9 for all samples Press Esc to return to the Life Science folder Press KHK to display available Options which are described below Version 1 0 Page 13 00 008 Version 1 0 Parameterz Print Graph Sample Humber Save Method Subo Frint Options select using key pad numbers 1 Return to parameters screen step 1 above 2 Print result via selected method 3 Toggle graph on off The graph shows a wavescan plot across the range 220 nm to 320 nm with cursors denoting 230 260 280 and if background correction selected 320 nm 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the alpha numeric keys to enter a name for the method and press Save 9 Auto print toggles auto print on off Exit options by pressing Ese or wait Page 14 2 RNA The procedure is as follows RHA Parameters Step 1 Press 2 to select RNA mode Select path length using the left and right arrows Options are 5 Dilution Factor Factor yl 10 el Step 3 dilution factor Known Enter the dilution factor using the keypad numbers Range 1 00 to 9999 Use the C button to backspace and clear
57. les until changed Step 12 standards selected Press replicates to display the replicate entry boxes Use C to clear previously stored results before measuring NA y F Insert the standard and press T to measure the standard and store the result Repeat for all replicates and standards Press Next to move from replicates of one standard to replicates of the next standard A graph will display the results and the fitted curve as the measurements are input Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 standards manual selected Press Next to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Page 37 sia bed Calibration Manual entry 0 050 4 E Shows previously entered calibration values and allows values to coro mm be entered via the keypad T 3 ye The highlighted box can be edited in order to enter an aoa v absorbance value corresponding to a given concentration value oso ME PA using the keypad numbers Range 0 001 to 9999 Use C to 0 1504 ra backspace and clear the last digit entered and the up and down E arrows to move between boxes mos Press OK to accept the calibration and go to the Results Da 04 m5 0 8 LO LE LY screen see below OR Press Back to return to the Standards screen Results screen Step 14 Insert th
58. ly performed Reagent test kits are routinely used for the enzymatic determination of compounds in food beverage and clinical laboratories by measuring NAD NADH conversion at 340 nm The change in absorbance over a specified time period can be used to provide useful information when an appropriate factor defined in the reagent kit protocol is applied Reaction rate and enzyme activity can be calculated if the factor used takes account of the absorbance difference per unit time as opposed to the absorbance difference per se For this reason the change in absorbance per minute AA min concentration AA min x factor and correlation coefficient calculated from a best fit of the data points are displayed They may not be relevant for simple kinetics experiments The procedure to define a new method is as follows Kinetics Parameters 1 Kinetics Parameter 1 Screen Step 1 Wavelength Wavelength Delay Time Enter the wavelength using the keypad numbers or the left and right arrows Range 190 1100 nm Press the down arrow Duration Step 2 Delay time Enter the delay time in seconds before measurements are taken This can be a maximum of 600 seconds 10 minutes Interval Press the down arrow Step 3 Duration Enter the time in minutes over which measurements are taken Cancel This can be a maximum of 60 minutes Press the down arrow Step 4 Interval Enter the interval time in seconds between measurements using the left a
59. measure the standard and Insert the standard and press store the result Repeat for all replicates and standards Use Next ES to bring up fields for the next standard A graph will display the results and the fitted curve as the measurements are input Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 standards manual selected Press OK to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Page 31 BCA Calibration Standards 0 200 0 400 0 600 0 200 1 000 1 400 0 051 4 0 171 A He 0 288 A PP 0 404 4 0 516 A f 0 627 A One vie Di na 0 4 0 5 18 10 lE 14 wavelength FBS nm Absorbance 0 2838 Curve Fit Regression 00 000 Version 1 1 Parameterz Print Graph Sample Miumber Save Method Subo Frint Calibration Manual entry Shows previously entered calibration values and allows values to be entered via the keypad The highlighted box can be edited in order to enter an absorbance value corresponding to a given concentration value using the keypad numbers Range 0 001 to 9999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Pressing the down arrow from the last standard will bring up the OK box Press OK to accept the calibration and go to th
60. nd right arrows Options are 1 5 10 20 30 or 60 seconds Press the down arrow Step 5 Press Next to go to the next parameters screen OR Press Cancel to return to the Applications Folder Kinetics Parameters 2 Mode Delta A Kinetics Parameters 2 Screen Step 6 Select the measurement mode using the left and right arrows Delta A change in absorbance over the measurement duration or selected period Final A absorbance at the end of the measurement duration or selected time Slope rate of change of absorbance over the measurement duration or selected period Factor 1 000 Version 1 1 Page 50 Kinetics Parameters Z Step 7 Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Options key Kk and then use the left right arrows ug ml ug ul pmol ul mg dl mmol l umol I g l mg l ug l U I ppm ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed decimal points DP to be selected from 0 to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen Esc parameters or Cancel Step 8 Set the Factor by which the result is multiplied to give the amount in the chosen range using the left and right ar
61. nly if the product has been used according to the instructions supplied General Electric Healthcare can accept no liability for loss or damage however caused arising from the faulty or incorrect use of this product e This product has been designed and manufactured for General Electric Healthcare by Biochrom Ltd 22 Cambridge Science Park Milton Road Cambridge CB4 OFJ UK However please contact General Electric Healthcare in the first instance if you experience technical or sample handling difficulties Version 1 0 Page 74
62. o a given concentration value using the keypad numbers Range 0 001 to 9999 Use C to backspace and clear the last digit entered and the up and down arrows to move between boxes Press OK to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Results screen Step 13 Insert the reference and press the 0A 100 T key This will be used for all subsequent samples until changed Step 14 Insert the sample and press FW The concentration of the sample is taken and displayed Repeat step 14 for all samples Press to return to the Applications Folder Dernee Wl ta Aianlays avianilahla Cntinne varhinh ara Ainanriharnd Version 1 1 Page 55 OOo 000 Parameters Print Graph Sample MHumber Save Method A2uto Print E Version 1 1 Options select using key pad numbers 1 2 3 7 Exit options by pressing Return to parameters screen step 1 above Print result via selected method Toggle graph on off Displays calibration graph cursors give values for last measured sample Sample number add a prefix to the sample number and reset the incrementing number to the desired value Save method use the left and right arrows to select a folder to store in Favourites Methods 1 9 press the down arrow and enter name Auto print toggles auto print on off Ese or wait Page 56 6 Multiple Wavelength This makes up to 5
63. o write text descriptions where appropriate or required Use repeated key presses to cycle through lower case number and upper case Leave for 1 second before entering next character Use C button to backspace and 1 to enter a space Escape from a selection and return to the previous folder Set reference to 0 000 A or 100 T on a reference solution at the current wavelength in the mode selected When in scan mode make a reference scan Enter or confirm a selection Take a measurement Page 6 ejolilololelolrie Parameters Print Print Data Set tO Ab Cursor Set tn At Cursor Slope Sample Humber Save Method Auto Frint s Version 1 0 Options select using key pad numbers i View parameters for the experiment 2 Print the results 3 4 5 6 Described in the application 7 Define the sample number you wish to start from 8 Save the parameters as a method to a defined folder name with a defined method name 9 Toggle auto print on off Default is off Exit options by pressing Esc or wait Experienced operators can use the numeric keys as a shortcut to the option required without needing to enter the Options menu Page 7 Software style The user interface is built around having folders of files which are displayed on the home page when the instrument is switched on Different folders are numbered and opened by using the associated number key on the keypad GeneQuant 1300 E E i 9
64. or all samples Press to return to the Applications folder Press KHK to display available Options which are described below Version 1 1 Page 45 Concentration Step 6 if using standard mode Insert the reference Press 0A 100 key This will be used for all o n subsequent samples until changed Concentration Press Ml to display the Run Standard screen l Run the standard by pressing 2 OR Press cancel Esc to return to the measure screen Concentration Step 7 Wavelength Insert the sample and press a l 260 nm 2 The concentration of the sample is displayed Results shown as indicate the concentration is out of range soca Repeat step 7 for all samples Press to return to the Applications Folder Press KHK to display available Options which are described yma below Options select using key pad numbers Return to parameters screen step 1 above E Parameters 2 Print result via selected method Print 3 Toggles on off displaying a graph of wavescan 20 nm Graph from selected wavelength C Fun Standard 4 Return to Run Standard screen 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value E Sample Number 8 Save method use the left and right arrows to select a folder Ed Save Method to store in Favourites Methods 1 9 press the down arrow E Auto Print wf and enter name 9 Auto print toggles auto prin
65. or optical density measurements of cell culture solutions to prevent the suspension settling too quickly and giving an OD that changes with time glycerol should be added to the sample The procedure is as follows OD 600 Parameters Step 1 Press 6 to select OD600 mode wavelength Step 2 Select the wavelength using either arrow or alphanumeric keys Default value is 600 nm E Press the down arrow 2 000 Step 3 Enter the correction factor to compensate for different optical configurations between this and other instruments Default value Is 2 Press the down arrow Step 4 Select the units Options are OD or cells ml If cells ml is selected two further parameters are displayed OD 600 Parameters Step 5 if cells ml selected rerun Enter the factor using the keypad numbers Range 0 00 to 9999 ee IC C button backspaces and clears the last digit entered Press the down arrow Correction Multiplier saan Step 6 if cells ml selected Select the multiplier using the left and right arrows Options are 1000 or 1 000 000 Step 7 Press OK to enter the Results screen OR Press Cancel Esc to cancel selections and return to the Life Science folder Version 1 0 Page 25 wavelength B00 nm Absorbance 0 0025 De 000 Version 1 0 Parameters Print Sample Humber Save Method Auto Print Results Screen Step 8 Insert the reference and press the 0A 100 T key This will be used for all sub
66. r to store in Favourites Methods 1 9 press the down arrow E Sample Humber and enter name E Save Method 9 Auto print toggles auto print on off El Awuto Print Ww E Eso Exit options by pressing Or wait Version 1 1 Page 57 7 Absorbance Ratio This makes simple absorbance ratio measurements on samples measuring the amount of light that has passed through a sample relative to a blank this can be air at two wavelengths The procedure is as follows Absorbance Ratio Wavelengths wavelength 1 wavelength 2 250 nm Background O m Absorbance Ratio Parameters Pathlenath Factor 1 000 Dilution Factor 1 000 Absorbance Ratio Parameters Dilution Factor olume Diluent 0 000 Version 1 1 Step 1 Enter the first wavelength by using the keypad numbers or the left and right arrows Press the down arrow Step 2 Enter the second wavelength as above Press the down arrow Step 3 Select whether a background correction is applied to both wavelengths 1 and 2 using the left and right arrows Step 4 If background correction is On Enter the third wavelength from which the background correction will be obtained Step 5 Press Next to enter the Parameters screen OR Press Cancel to return to the Applications Folder Absorbance Ratio Parameters Screen Step 6 Select the pathlength 5 or 10 mm using the left and right arrows Press the down arrow
67. r wait Peak Detection Shortcut button 4 AutoDetect Peaks Turns on and off the automatic peak detection The following options determine how peaks are detected Minimum peak height Minimum height the peak has to be above the higher of the two adjacent minima for the peak to be detected Minimum peak width Minimum width of the peak as determined by the difference in wavelength between the higher of the two adjacent minima and the opposing intersection of that higher minimum level and the peak profile See the screen displayed below Peak Detect on Zoom Determines whether peaks are re assessed and tabulated when the user zooms into a region of the wavescan If off leaves the peak detection as determined on the un zoomed display Sort peaks by Determines the sequence that peaks are reported by Can be wavelength peak height or peak width Draw Peaks Switches display of peak cursors on and off These show vertical dashed lines displaying the measured peak height and horizontal dashed lines showing the peak width Pressing Cancel Eso ignores the selection pressing OK accepts them Page 48 W avescan ae User eens Peak Add Peak Shortcut button 5 I 0 4 Adds a used defined peak at the current cursor position The aa i entry is then displayed in inverse colouring to discriminate A pa i between user defined peaks and auto detect peaks When the m e cursor is positioned over the user defined peak
68. rint Exit options by pressing F or wait Version 1 1 Page 41 THE APPLICATIONS FOLDER Applications o e Single wavelength Concentration Ma avescamn oD eG o Kinetics Standard Cure Multi wavelength Pla o Absorbance Ratio SUMMARY Function Key pad number Description o denia 1 Absorbance or T transmission at a single user defined Single wavelength wavelength i N e Concentration measurement at a single wavelength based on a Concentration simple Factor entered or calculated from a single standard o 3 Wavelength scan between two user defined wavelengths Range Wlavescan 200 900 nm with user configurable peak finding function o 4 Absorbance versus time measurements either rate or end value kinetics based o 5 Generation of calibration curve by measuring standards at a single Standard Curve wavelength mm o 6 Absorbance or T transmission at up to 5 user defined Multi wavelength wavelengths Fiaa se O 7 Ratio of absorbance values at two user specified wavelengths Absorbance Ratio OPTIONS Within each application the user has the possibility to select various options that define the way results are treated If not using a stored method it is advisable to check that these Options have been appropriately set for your experiment when coming to the instrument Note that setting the History parameter to on see Preferences later will cause the instrument to store ts last settings If the
69. rows Range of 0 01 to 9999 Step 9 Press OK to enter the Results screen OR Press Back to return to the Parameters 1 screen Results Kinetics Insert the reference and press the 0A 100 T key i Cpr see ee ha pe Insert the sample and press E i to start the run Time min is displayed at the bottom of the screen and absorbance data are plotted on the graph as testing proceeds The table below the graph gives absorbance values at T start Ms of calculation T finish of calculation change in absorbance slope regression parameter R of the calculated slope and the 0 0 0 4 E Bo An da Slope MEA It calculated from the selected ter dA final A aoe aate ones oro ow EN Sample 1 Time 00 00 10 Abs 0 231 0 6 Use the left and right arrows to move the cursor and display the time and absorbance value at measured data points Use the up and down arrows to zoom in or out Press Cancel to return to the Applications Folder Press KHK to display available Options which are described below Version 1 1 Page 51 O a G O O O Version 1 1 Parameters Print Print O ata Set tO At Cursor Set tn At Cursor Slope Sample Humber Save Method Auto Print Options select using key pad numbers 1 2 3 4 Exit options by pressing Return to parameter 1 screen step 1 above Print data on the results screen via selected method Print all the
70. sequent samples until changed Step 9 Insert the sample and press l The wavelength absorbance and OD600 value is displayed Repeat step 9 for all samples Press to return to the Life Science folder Press KHM to display available Options which are described below Options select using key pad numbers Return to parameters screen step 1 above 2 Print result via selected method 7 Sample number add a prefix to the sample number and reset the incrementing number to the desired value 8 Save method use the alpha numeric keys to enter a name for the method and press Save 9 Auto print toggles auto print on off Esc Exit options by pressing Or wait Page 26 7 Protein Determination The GeneQuant 1300 includes five methods for assaying proteins Press 7 to enter the protein folder Life Science Protein o ef ef BCA Protein LU Bradford of ef Lown Biuret Protein Determination at 280 nm Protein can be determined in the near UV at 280 nm due to absorption by tyrosine tryptophan and phenylalanine amino acids Abs 280 varies greatly for different proteins due to their amino acid content and consequently the specific absorption value for a particular protein must be determined The presence of nucleic acid in the protein solution can have a significant effect due to strong nucleotide absorbance at 280 nm This can be compensated by measuring Abs 260 and applying the equ
71. settings and return to the Utilities folder OR Press Cancel to return to the Utilities folder without storing the settings Version 1 0 Page 62 4 Preferences Sets user preferences The procedure is as follows Preferences Select games function This determines whether the games Games Auto Standby folder is displayed or not Options are yes or no or Press the down arrow Define the screen layout of folders Options are either a grid Theme format default or a list Press the down arrow Select whether to use previously entered parameters when the History instrument is switched on or to use defaults he Sie Press the down arrow Select whether to use a standby mode after defined periods Options are 1 hour 2 hours at night or off Press OK to store the settings and return to the Utilities folder OR Press Cancel to return to the Utilities folder without storing the settings 5 Contrast Contrast Contrast Adjust the contrast using the left and right arrows Press the down arrow ae Adjust the brightness using the left and right arrows dicii Press the down arrow Press OK to store the settings and return to the Utilities folder Ambient temperature can affect the display This function can optimise the display for local conditions The procedure is as follows 6 Folder Names Folder Names Select the folder you wish to rename using the left and right arrows Press t
72. ss Save CP Save Method 9 Auto print toggles auto print on off El Auto Print yA Esc Exit options by pressing Or wait Version 1 0 Page 22 5 Cy dye Measurement of the labelling efficiency of fluorescently labelled cDNA probes before 2 colour microarray hybridisation ensures that there is sufficient amount of each probe to give satisfactory hybridisation signals These data also provides an opportunity to balance the relative intensities of each fluorescent dye by adjusting the concentration of each probe before hybridisation The cDNA yield is measured at 260 nm while the incorporation of fluorescein Cy3 and Cy5 are measured at their absorption peaks This method may also be useful for measuring the yields and brightness of fluorescently labelled in situ hybridisation probes Cy Oye Parameters Dye Name Pathlength wavelength Background on Coefficient 0 750 Cy Dye Settings Dilution Factor Start ANA Fl 1 000 1000 Yolume II Cy Dye Settings Volume Cy Dye Settings Dilution Factor Start AWA 1 000 1000 Factor Background Volume Version 1 0 Step 1 Press 5 to enter the Cy dye parameter screen Step 2 Enter the name of the dye being used the default is Cy3 but others may be generated using the Clear key C and the alphanumeric keypad Press the down arrow Step 3 Enter the wavelength of the dye peak absorbance Press the down arrow Step 4 Enter the dye coeffic
73. standards manual selected When all standards are measured the OK box appears Press OK to accept the calibration and go to the Results screen see below OR Press Back to cancel selections and return to the Standards screen Calibration Screen replicates on This shows the calibration values and allows standards to be measured Step 11 standards selected Insert the reference sample Press 0A 100 key This will be used for all subsequent samples until changed Step 12 standards selected Press replicates E to display the replicate entry boxes Use C to clear previously stored results before measuring ah Insert the standard and press M to measure the standard and store the result Repeat for all replicates and standards Press Next to move from replicates of one standard to replicates of the next standard A graph will display the results and the fitted curve as the measurements are input Use the up and down arrows to select a standard to be repeated if a poor reading has been obtained Use C to clear the previous reading Step 13 standards manual selected Press OK to accept the calibration and go to the Results screen see below OR Press Back to return to the Standards screen Page 40 Biuret Calibration Calibration Manual entry 3 Shows previously entered calibration values and allows values to 0 5 0 400 0 145 A 0 600 002494 ay 0 800 0 345 A 0 3 0 2 be entered via
74. t on off Exit options by pressing F or wait Version 1 1 Page 46 3 Wavescan An absorption spectrum can be obtained from your instrument enabling simple identification of peak height and position The procedure is as follows Wavescan Parameters Start wavelength End wavelength Mode Ok 420 440 460 480 Version 1 1 Step 1 Set start wavelength by using keypad numbers or left and right arrows Range 200 890 nm Press the down arrow key Step 2 Set end wavelength by using keypad numbers or left and right arrows Range 210 900 nm Press the down arrow key Step 3 Select the mode Absorbance or T using the left and right arrows Step 4 To enter the measurements screen with the selected parameters press OK 7 OR Cancel the selections and return to the Applications Folder by pressing Cancel E Step 5 Insert the reference Press 0A 100 key This will be used for all subsequent samples until changed Step 6 Insert sample and press a Repeat step 6 for all samples Results A graph of the wavescan is displayed along with a table of Absorbance T at each peak Use the left and right arrows to move the cursor along the graph When it reaches a peak the peak height and width of the peak is displayed at the top of the screen To zoom in on the wavelength scale use the up arrow This auto scales on the Absorbance T scale dependent on the Graph Scale option and this is
75. th file format and connection e PVC can save data in graphics format text format or as an Excel file Installation See the manual included on the PVC CDROM for installation and operating instructions Version 1 0 Page 72 ACCESSORIES USB cable source locally Built in printer accessory 80 3003 84 Bluetooth accessory 80 3003 96 MAINTENANCE After Sales Support Support agreements that help you to fulfil the demands of regulatory guidelines concerning GLP GMP are available e Calibration certification using filters traceable to international standards e Certificated engineers and calibrated test equipment e Approved to ISO 9001 standard Choice of agreement apart from break down coverage can include e Preventative maintenance e Certification When using calibration standard filters insert such that the flat surface is facing away from the spring end of the cell holder Observe all necessary precautions if dealing with hazardous samples or solvents Lamp Replacement The xenon lamp should not need replacement until after several years of use In the unlikely event that it does need replacing this should be undertaken by a service engineer from your supplier Cleaning and general care of the instrument External cleaning Switch off the instrument and disconnect the power cord Use a soft damp cloth Clean all external surfaces A mild liquid detergent may be used to remove stubborn marks Changing cell holder or removal
76. the desired value 8 Save method use the alpha numeric keys to enter a name for the method and press Save 9 Auto print toggles auto print on off Esc Exit options by pressing Or wait Page 29 2 BCA The procedure is as follows Step 1 Press 2 to select BCA mode Step 2 Curve Fit The wavelength for this method is set at 562 nm tLe Enter the number of standard concentration points 1 9 to be TEE Calibration used in the curve using the keypad numbers or left and right Press the down arrow Units Replicates BCA Parameters Step 4 Units The user can enter a text string up to 8 characters long To access a list of pre defined units press the Options key II and then use the left right arrows ug ml ug pl pmol ul mg dl mmol l umol l g l mg l ug l U I ppm AA ppb conc or none These units can also be edited once OK is pressed This screen also allows the number of displayed decimal points DP to be selected from 0 to 2 Note that the result will always be fixed to 5 significant figures regardless of how many decimal points are selected so 98768 2 will display as 98768 even with 1 decimal point selected Press OK to store the chosen parameters or Cancel Esc Step 5 Enter the type of curve fit Options are straight line regression zero regression forces the straight line through the origin interpolated or cubic spline Press the down arrow BCA Parameters Step 6 Sel
77. the last digit entered OR Step 3 calculate dilution factor Press XII to enter the dilution factor screen see second image to the left Enter the volume of the sample using the keypad numbers Range 0 01 to 9999 Press the down arrow Enter the volume of the diluent using the keypad numbers Range 0 01 to 9999 Press OK to calculate the dilution factor and return to the Parameters screen RHA Parameters OR Press to cancel the selections and return to the Parameters screen Step 4 Select whether the background correction at 320 nm is used or not with the left and right arrows Press the down arrow Step 5 Select the units of measurement using the left and right arrows Options ug ml ng ul ug pl Press the down arrow Step 6 Enter the factor using the keypad numbers Default value is 40 range is 0 01 to 9999 Step 7 Press OK to enter the Results screen and start taking measurements OR to return to the Life Science folder Results Screen Step 8 Insert the reference sample Press 0A 100 T Key This will be used for all subsequent samples until changed Step 9 Insert sample and press B This measures at the selected wavelengths and displays the results The ratio of wavelengths 1 and 2 absorbencies are calculated both corrected by the background wavelength value if selected Gives concentration based on absorbance at wavelength 1 Repeat step 9 for all samples Press
78. used configured methods Methods Contains nine folders that can store less frequently used configured methods nine methods per folder Utilities Instrument set up date time language etc and games The instrument is supplied with a program PVC Print via Computer on the accompanying CD When used with a USB cable to connect to a PC onto which the software has been installed it enables the user to print through the PC directly to the printer that is connected to it The data may also be stored as an Excel spreadsheet as an EMF graphics file a comma delimited csv data file a tab delimited txt data file or in native PVC format for later access Alternatively results may be sent to the PC via a Bluetooth accessory this can either be supplied pre installed or is available as an optional accessory if the need for its use arises after installation of the product PVC works in a similar way A printer is available for the instrument this may either be supplied pre installed or is available as an optional accessory if the need for its use arises after installation of the product Sample handling tips e Note that the light beam is directed from RIGHT to LEFT through the cell chamber therefore please ensure the cell is inserted in the correct alignment e The cell holder supplied with the instrument accepts standard 10 mm pathlength quartz glass or plastic cells e The optical height is 15 mm and the minimum volume that can be use
79. wn arrow Dilution Factor s Enter the volume of the diluent using the keypad numbers Range 0 01 to 9999 Press OK to calculate the dilution factor and return to the Parameters screen Oligo Parameters Yolume Diluent OR Press to cancel the selections and return to the Parameters screen Step 4 Select whether the background correction at 320 nm is used or not with the left and right arrows Press the down arrow Step 5 Select the units of measurement using the left and right arrows Options ug ml ng ul ug l and pmol ul If pmol ul is selected the Oligo Parameters factor changes to a selection table denoting the ratios of the 4 bases in the structure Pathlenath Press the down arrow 10 rm Step 6 units not pmol ul Enter the factor using the keypad numbers Default value is 33 Dilution Factor range is 0 01 to 9999 1 000 OR Step 6 units pmol ul Enter the proportions of bases present using the keypad numbers and up and down arrows to move between boxes Default is 10 for each range is 0 to 9999 Background a Step 7 Press OK to enter the Results screen and start taking measurements OR ESC to return to the Life Science folder Version 1 0 Page 17 Results Screen Step 8 Insert the reference sample Press 0A 100 T Key This will be used for all subsequent samples until changed Step 9 Insert sample and press E This measures at the selected wavelengths and displays the results T
80. zing using a multiplying factor of 100 because of their greater binding power Theoretical Absorbance The Theoretical Absorbance is based on a calculation as follows For each adjacent pair of bases nearest neighbours an extinction coefficient weight is accumulated using a 4x4 table one for either DNA or RNA This total weight is then doubled Then for each internal base a counterweight is subtracted using another 1x 4 table The end bases are excluded from the latter summation That is Total Extinction Coefficient E x 2 x alable base_type base n base n 1 tTable base_type base n Version 1 0 Page 19 Conversion factor The Conversion Factor is given by Molecular weight agcpe 2 Eascoe where Eascoe 2 x Eas Egc Eco Epe Es Ec Ebp e The molecular weight MW of a DNA oligonucleotide is calculated from MW g mole dA x 312 2 dC x 288 2 dG x 328 2 dT x 303 2 MW counter ion x length of oligo in bases for RNA oligonucleotide dT x 303 2 is replaced by dU x 298 2 The MW calculated using this equation must be adjusted for the contribution of the atoms at the 5 and 3 ends of the oligo For phosphorylated oligos add 17 2 x MW of the counter ion For non phosphorylated oligos subtract 61 MW of the counter ion The MW g mole of the most common oligo counter ions are Na sodium 23 0 K potassium 39 1 TEA triethylammonium 102 2 Other 1 Ca

GeneQuant 1300 User Manual - GE Healthcare Life Sciences

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