The computer controlling the plate reader runs Windows XP. Some familiarity with Windows should be sufficient to use it.<\/p>\n
Accounts on computer<\/p>\n
<\/p>\n
Data can be moved from the computer by writing it on a flash drive, email or CD.<\/p>\n
Start SoftMax Pro using the icon on the Quick Launch toolbar or on the desktop.<\/p>\n
There should be an icon for the manual on the desktop. Your files should be automatically saved to the My Documents<\/b> folder with the autosave feature. In addition, when you manually save data, (File-save as<\/b>) it should be in the My Documents<\/b> folder until you change it.<\/p>\n
After starting the program, you should find a list of protocols on the Assay<\/b> menu. The list is a standard grouping supplied by Molecular Devices and includes some which cannot be used on this instrument; they can be deleted. You can add your own protocols.<\/p>\n
You can save new protocols.<\/b> Protocols from other labs will be inaccessible; this is to reduce the size of the protocols list. When you save a data or protocol file, the folder used will be the last folder used, which normally should be your My Documents<\/b> folder. The program as delivered used the Program files-Softmax Pro folder. You can read files from that folder, but not store files there.<\/p>\n
If SoftMax Pro does not start, it is probably because someone else left it running. The simplest solution is to restart the computer (Start <\/b>button, Turn off computer<\/b> then restart<\/b>. Ignore the messages about others risking losing data and say yes<\/b> to restart.) After using the plate reader, close SoftMax Pro, and turn off the plate reader. Turn off the computer and display.<\/p>\n
If the computer does something strange so that restarting it seems the appropriate response, press power button and keep it depressed until the computer switches off; this will take several seconds.<\/p>\n
The instrument is a dual monochromator fluorescence instrument. This design enables it to read a wide range of fluorescent compounds. It cannot read absorbance.<\/p>\n
Wavelength ranges: 250 nm to 850 nm, excitation and emission, 9 nm bandwidth.<\/p>\n
Sensitivity (top read, signal 3X standard deviation of baseline) 3 fmole FITC in a 200 \u00b5L well; bottom read, 8 fmole of FITC<\/p>\n
No capability for polarized fluorescence<\/p>\n
Time resolved fluorescence-<\/b> a secondary mode<\/p>\n
Data collection: 50-1450 \u03bcsec., 200 \u03bcsec increments<\/p>\n
Sensitivity: 0.5 fmol\/well Eu-chelate (obtained with DELFIA\u00ae reagent from Perkin Elmer)<\/p>\n
Detection limit: 10 amol\/well alkaline phosphatase 200 \u03bcL\/well (obtained with Emerald IITM<\/sup> reagent from Applied Biosystems).<\/p>\n Because there are no injectors, it cannot perform flash luminescence like the Promega Dual Glo Luciferase assay.<\/p>\n The limited control over read time limits sensitivity.<\/p>\n Note that plates exposed to light may emit light for some time, increasing background.<\/p>\n Strips of wells should be avoided because they carry a risk of the strip falling into the machine requiring an expensive, time consuming repair by the manufacturer. If they must be used, rigid frames are recommended, and the strips must be securely installed.<\/p>\n The instrument takes several minutes to perform start up tests.<\/p>\n After the instrument is ready for use, and you have started SoftMax Pro, a window appears showing a plate and a setup<\/b> icon. Suggestions and explanations for the options are listed in the SOFTmax Pro manual p. 269:<\/p>\n To set the instrument for your reading, you can look for an existing method under the Assays<\/b> menu item, or enter setup\u00a0 by pressing the setup<\/b> button in the window, or double click the grey area with setting, or on the top menu go to Control-Instrument Setup<\/b><\/p>\n Fluorescence is the default. Top reading gives greater sensitivity for 96 well plates, unless you have cells adhering to the bottom of the well.(i.e. leave bottom read<\/b> box blank) Either select from drop down list or type in the wavelength you want for excitation and emission. The auto cutoff <\/b>box sets filters; leaving it on is suggested except for scans. Note that more than one wavelength can be read.<\/p>\n The photomultiplier tube setting can be changed for samples with high or low concentrations. The voltage on the tube is changed. This is equivalent to gain.<\/p>\n The number or readings can be increased to increase accuracy, at the expense of time. Maximum number of readings is 30.<\/p>\n This setting is to shake the plate. Leave off unless you know you need it.<\/p>\n This is an instrument calibration and should be done at least once per session. Omission of autocalibrations reduces time for readings.<\/p>\n a list of plates specifically supported shows, 96, 384, 24, 12, 6 well types. Use standard plate unless you see your type listed. Suggested plates are black walled. Clear plates will give more cross talk between wells.<\/p>\n Dark blue indicates well will be read. Part of a plate can be selected so that only a selected area of the plate is read.<\/p>\n Used if you define different plate sections; see p. 39 of SOFTmax PRO manual.<\/p>\n You can save your settings as a protocol under File-Save As<\/b>. In the dialog box, for file type, choose Protocol (ppr)<\/b>. This protocol should now appear in a list under the Assays<\/b> menu.<\/p>\n A saved assay protocol will put in settings for wavelength.<\/p>\n Temperature can be set to 4\u00ba above ambient to 45\u00b0C (thermometer icon) on main toolbar<\/p>\n The plate can be shaken (icon right of thermometer, and see automix option in setup).<\/p>\n Unless reading plates from the bottom, put plates on the purple adapter to put them closer to the reading head. Remove plate cover.<\/p>\n Press read<\/b> button on menu on top of page.<\/p>\n The manual for the plate reader describes how to determine the optimal wavelength settings for a fluorophore. If excitation and emission maxima are less than 80 nm different, see the documents below on how to find the best wavelength settings.<\/p>\n Alexa dyes are one class of compound that need careful choice of wavelengths. See documents: If you have two fluorophores, you may have better separation of the two signals by using non-peak wavelengths.<\/p>\n A few times the instrument did not work, because the wrong instrument selected in software. After the instrument has completed startup checks, in the top left of the computer screen, just under File<\/b>, there is an instrument icon. If there is a large red X after the instrument display says the instrument is ready, there is a problem.<\/p>\n Click on the icon to bring up a Preferences<\/b> window. On the left side of the window, near the top there is a box for serial port<\/b>. It should be COM1<\/b>. This information seems to be saved in the folder for protocols, so will not be saved if the folders are stored in the read only folder Program Files SoftMax Pro 4.7.<\/p>\n Note that a versatile monochromator based instrument like the Gemini EM is less sensitive than one using filters; the advantage is that it is easy to set the Gemini for a wide range of compounds.<\/p>\n Standard things to check if the sensitivity is low:<\/p>\n Top read setting?<\/p>\n Use of purple plate adaptor for top read<\/p>\n Is there something colored in the sample which absorbs light?<\/p>\n Try setting PMT to high in assay method<\/p>\n Increase number of readings to 30 in assay method to reduce noise<\/p>\n Optimize wavelengths-protocols from Molecular Devices are on computer desktop<\/p>\n Use all black plate<\/p>\n Consider non binding surface type plate (Greiner and Corning are 2 manufacturers)<\/p>\n Note that the instrument self check should detect a weak lamp.<\/p>\n Click template<\/b> button. A new window should appear. To designate wells as blank, standards or unknowns, click ontemplate<\/b>. Then select wells, and then click on the dropdown list next to group<\/b> in the top left corner to select the well type. If the well type is something other than blank, click new<\/b>. After drawing up a template, you can save it. One method is under the Plate<\/b> menu at the top of the screen, chose Export template. To use the template, go to Plate-import template.<\/b> Then select cells, and click the Group<\/b> button, and select type of sample from the drop down list. The wells should then have a label. Note the series button, useful if you have standards that increase in a fixed step.<\/p>\n There is an autosave feature with a default name. An addition, data can be manually saved so that you can give the file a name which helps identify the data.<\/p>\n Data can be saved in a format recognized by Excel;go to The default is to export data in a plate format. Excel normally recognizes how to open the file. Data is saved in .pda<\/b> files.<\/p>\n To get data from the computer, the choices are:<\/p>\n Printing<\/p>\n Writing to a CD<\/p>\n Writing to a USB flash drive<\/p>\n Many people export the data to a text file (File-export<\/b>. File type text<\/b>) and then analyze in Excel.<\/p>\n The SoftMax program can be installed on a limited number of other computers; the serial number of the software is needed, the instrument serial number is irrelevant.<\/p>\n Near the instrument is a book from Molecular Probes with advice on reading fluorescent samples. Life Science Technical Sales Representative<\/p>\n Molecular Devices Corporation<\/p>\n 800.635.5577<\/p>\nPlate types<\/h4>\n
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Operation<\/h4>\n
Type of data<\/h4>\n
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Read type<\/h4>\n
\nThe instrument has some capacity for luminescence readings, but does not have the capabilities of a dedicated luminometer. The instrument cannot perform a dual luciferase assay (e.g. Promega).
\nTime resolved fluorescence is for compounds which emit light after the lamp is off; this mode reduces background noise.<\/p>\nWavelengths<\/h4>\n
Sensitivity<\/h4>\n
Automix<\/h4>\n
Autocalibrate<\/h4>\n
Assay plate type<\/h4>\n
Wells to read<\/h4>\n
Auto read<\/h4>\n
Other features<\/h4>\n
Wavelength settings<\/h4>\n
\n30-Wavelength Selection on Gemini
\n<\/b>31-Wavelength Optimizing on Gemini
\n<\/b>on the desktop of the user accounts.<\/p>\nFor some common wavelengths, see<\/h4>\n
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Problems<\/h4>\n
Instrument does not work.<\/h5>\n
\nIn the lower half of the window, left side, there is a box labeled reader<\/b>. The entry should be SpectraMax GEMINI EM<\/b>. If it is something different, click on the arrow to bring up a list of instruments, and select the correct entry.<\/p>\nLow sensitivity<\/h4>\n
Setting standards, unknowns, dilutions<\/h2>\n
\nIn the group settings window, click on list under group format.
\nFor unknowns, you can enter a dilution series.
\nFor standards, select cells, click the list for group<\/b>, select new<\/b>, then in the group settings window, under column format, select standards. If you click the sample descriptor<\/b> box, you can chose the name for the standards and units.<\/p>\nData-saving and moving<\/h4>\n
\nFile-export<\/b>.
\nFor file type, choose text.<\/p>\n
\nTo export in time format, go to Edit-preferences.<\/b> In the left side of the window is a box for data export format, either time or plate. Time format is useful for kinetic data.<\/p>\nSoftware-SoftMaxPro-data analysis<\/h4>\n
Help<\/h4>\n
\nThe software manual is also present, and on the computer desktop.<\/p>\n