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1. P Figure 7 3 Lane Volume Excision Tool image with lanes 3 Click Stretch Frame i and then click and drag the frame anchors to fit the frame over the gel Optional Make additional adjustments to the lane frame as needed e Click Add Adjust Anchors SN and then click on the top or bottom of the gel to add new anchors Click and drag the lane anchors to adjust them e Click Remove Anchors py and then click on a frame anchor to remove it e Click Create Lane le and the do a click and drag to draw a new lane between existing lanes K e Click Adjust Lane x and then click and drag a lane to move it or divide it into multiple segments Dividing a lane into segments helps to account for curvature in the lane o Click Unadjust Lane E and then click on the lane segment markers to remove them Note that if only one segment is present then the lane is removed o Click Remove Lane fis to delete a lane 5 Adjust the Vertical slider to control the vertical cut spacing Use the decrease tool to see the effect of changing the vertical control Increase the vertical spacing to decreases the number of cuts e a S MS ad Tr e au m A ee Figure 7 4 The effect of the Vertical control on the cut requests Left Vertical 0 Right Vertical 100 LANE VOLUME EXCISION TOOL 6 Adjust Horizontal slider to control the horizontal cut spacing Use the zoom tool to see the effect of changing the Horizontal control a no k pa en
2. Void the EN 61010 1 safety certification Create a potential safety hazard Bio Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than those for which it is intended or by modifications of the instrument not performed by Bio Rad or an authorized agent EN61010 1 is an internationally accepted electrical safety standard for laboratory instruments DI INTRODUCTION 1 0 INTRODUCTION 1 1 OVERVIEW The EXQuest spot cutter is a precision instrument that accurately locates and excises protein spots and bands from one and two dimensional gels or blots It then loads them into microplates for down stream processing and analysis It is designed to cut spots and bands from gels and blots with high efficiency and then deliver them to 96 and 384 well microplates or 96 tube racks The EXQuest spot cutter is designed to be both robust in construction and simple to operate It has the capability to image gels and blots that are visibly stained e g silver and Coomassie and fluorescently stained e g SYPRO Ruby and SYPRO Orange Figure 1 1 EXQuest Spot Cutter 1 1 INTRODUCTION 1 2 SYSTEM DESCRIPTION The EXQuest spot cutter is a fully integrated system controlled through PDQuest and Quantity One software lt consists of an enclosure an imaging system a fluidics system robotics sensors a cutting head a gel tray a microplate rack and a wash station A det
3. WW Mass Spec Score Overlay Figure 6 21 Identify pull down menu In the Select Analysis Set panel of the Analysis Set Excision Tool window click the Name down arrow and select the desired analysis set The number of spots contained in the analysis set will be displayed in the Spots field Step 1 Select Analysis Set Name Sample Analysis Set 7 Spots 204 Figure 6 22 Step 1 Select Analysis Set panel Click the Step 2 Select Gel Setup tab and identify which gel s are to be cut This is accomplished in one of two ways Select from list Choose the Select from list option and then select one or more gels to cut Multiple gels may be selected by Ctrl click or Shift click In the list the Spots column lists the number of spots for each gel in the analysis set and the Cuts column shows the number of cuts that have been selected from each gel In this mode all the spots in a selected gel will be cut unless they are deselected on the Review tab Check the Place cut of same spot on different gels into same well Pool cuts box to pool spots with the same Standard Spot SSP number but cut from different gels ANALYSIS SET EXCISION TOOL Step 2 Select Gels Gel Spots Cuts Select from list Ki sample3 vi 12 0 sample vi 204 204 C Autoseleci Ki sample v2 52 0 Gels 1 Figure 6 23 Step 2 Select Gels Select from List Auto select a Choose Auto select to let PDQuest automatically select the b
4. T EXQuest Spot Cutter Setup xj Plate Position Calibration Would you like to calibuate the positioning of another type of container dnjas sanox3 Back Next gt Cancel Figure 3 12 Plate position calibration calibrate another microplate format 11 Wash Station Position Calibration The wash station position calibration step is used to center the cutting tip over the wash ports a Use the Left Right and Front Back slider to center the cutting tip over the wash port The gel tip is centered over the left wash port see Figure 3 13 and the membrane tip is centered over the microtube in the left membrane tip wash port Click on the slider bar to move the tip in small increments or click and drag the slider to make larger adjustments When the tip is centered check Lower head to adjust eject height to lower the cutting tip into the well Use the sliders to center the tip into the well EXQUEST CALIBRATION WIZARD 3 EXQuest Spot Cutter Setup xj Wash Station Position Calibration Position the head over the wash station as shown It should be centered in the well and the tip when lowered dnjas isanox3 Figure 3 13 Wash station position calibration b When the tip is centered check Lower head to adjust wash height to lower the cutting tip into the well Use the sliders to center the tip in the well c Use the Raise Lower slider to adjust the tip height Typically the gel tip should
5. Figure 2 11 Software license screen 2 To start the software in Basic mode click Basic 3 To run the fully featured version make sure your HASP key is connected to a USB port before you start the software EXQUEST CALIBRATION WIZARD 3 0 EXQUEST CALIBRATION WIZARD Before you can use the spot cutter for the first time you must calibrate it The calibration wizard helps you to quickly calibrate the camera microplate and wash station The calibration wizard functions the same whether you are calibrating the gel head or the membrane head Calibration should be performed when the instrument is set up for the first time and then repeated whenever the cutting head is changed new fluorescent or halogen lights are installed the camera is refocused the spot cutter is moved or optimum accuracy is required It is recommended that the EXQuest spot cutter be recalibrated at least once every three months To calibrate the spot cutter 1 Start the PDQuest or Quantity One software and click Edit Preferences File view Image Spots Text Overlays x Calibrate Gels F Preferences Colors Mouse Mouse Macros F Figure 3 1 Edit pull down menu 2 Click on the Devices tab in the Preferences dialog D Preferences q Mec Paths Display Toobars Application Devices Securty imagers Spot cutter Select the Imagers to be diplayed in the fle menu C Not set x Gel Doc EQ 5
6. Prepare the gel blot as described in Section 4 Open the Quantity One software and click Excision Lane Volume Excision Tool is Excision Reports Window Help Lane Wolume Excision Tool a HO A B Manual Excision Tool Figure 7 1 Excision pull down menu 4 Make sure that the DI water bottle is full and the waste bottle is empty at the start of the run and click Prime Pump 5 If cutting blots fill three 1 5 ml capless microtubes with water to approximately 1 mm below the tube top and then place the tubes in the three membrane tip wash wells After making a cut the cutting tip will be dipped into one of the three tubes after each cut to wash the tip and remove any membrane plugs that have adhered to the tip 6 Inthe Acquire Image panel see Section 8 1 select White Trans for visible stained gels such as Coomassie or silver stain or UV EPI for fluorescent stains such as SYPRO Ruby 7 Close the spot cutter front door and click Acquire Image to take an image of the gel tray contents Do not open the door during image acquisition As a safety precaution the UV EPI lights will automatically turn off if the door is opened Images acquired with UV EPI are automatically inverted by the software to display dark spots with a light background If the image does not detect enough spots or is too faint use the transform function to optimize the image contrast see Section 8 3 8 Identify the cutting tip size 1 0 mm or
7. EXQuest Spot Cutter Instruction Manual Catalog numbers 165 7200 EXQuest Spot Cutter 165 7201 EXQuest Spot Cutter With Computer On the Web at http www bio rad com For Technical Service Call Your Local Bio Rad Office or in the US Call 1 800 4BIORAD 1 800 424 6723 TABLE OF CONTENTS TABLE OF CONTENTS SAFETY SECTION 1 INTRODUCTION ocnccccccoccccnncnnnannnnnnrnnnnn rc 1 1 DS AE A EE he casty uss ccadeeartodcasaenstc casks E A A E E niet 1 1 1 2 System Descripto M sais cei Ae ei i aia dee ee 1 2 1 2 1 Gel Imaging System cnmcccinnnncnnncccnnnnnnnnnnccnnnrnn naar 1 2 1 22 Imaging System EE 1 3 1 2 3 Gel Tray and Gel Clamps eeccccccccceceseceeeeeeeeeaeeeeeeeeceaaeseeaeeesaeeseeaaeesseeeteaeeesnees 1 3 L Gutting HO ad aiii Ad a vee eee di 1 4 1 25 Fluidics System ita iria 1 5 1 2 6 Wash Sollana tds 1 5 LS Microplate Ra oi eege ett e EE Se NEE ebe e EES 1 6 1 28 eelere 17 RS A edt lee eet ae eee 1 8 1 2 10 Front Panel Controls and Indicators eccccccceeeeeeeeececeeeeeeeeeeeeeeeaaeeseneeeseaeeneneees 1 8 1 211 SOMWANE Sz A EE AOE E 1 9 1 2 12 Barcode Rede Tico ii saueebavedaectenvebenetivecegeniveeedeeeei 1 9 SECTION 2 SYSTEM INSTALLATION cccceccsseeeeseeeeeeeeeesnneeeneeeeesaeeenseeeessaaesaseeeeseessaneeeeeseeeeaneaeeeeenaes 2 1 21 edu EI 2 1 2 2 Unpacking the EXQuest Spot Cutter ooococcccnnocicinccccononanonccccnnnnnnn ano nn cnn n canaria cerraran 2 1 2 3 System Leveling cta
8. L am P se 140 Ae a aie _ gt wre em EMM an aie E i W i gt AN emm W Se sg S em GE e e Les ua 1507 al EA ft a Figure 7 7 Lane Volume Excision Tool Display Excisions Review the Run Info panel see Section 8 8 for the number of cuts selected the number of plates required and the required plate well volume Click Begin Resume to open the Plates for Cut Run dialog see Section 8 9 and then enter the plate name and barcode plate ID in their respective fields The plate barcode can be entered using a barcode scanner or typed in To reserve wells on a microplate click the Edit button for the desire plate and click on individual wells to reserve them Press Done to open the Load Plates on Cutter dialog box see Section 8 10 Locate the microplate with the name and barcode plate ID shown in the dialog and then scan or type in the barcode plate ID into the Confirm barcode field If the Confirm barcode does not match the Barcode a warning will pop up that states there is a barcode mismatch In this case make sure that you have the correct microplate and have entered the barcode correctly Place the plate in the microplate rack position identified by the green box with well A1 located in the front left plate corner Click Done to proceed to the next plate or Prev to return to the previous plate Click Finish to start the run If at any time the cut run needs to be paused o
9. see Section 8 5 and then specify the well volume and well loading order The well volume will determine the maximum number of spots that can be placed in a single well tube Review the Run Info panel see Section 8 8 for the number of cuts selected the number of plates required and the required plate well volume Click Finish to finish creating the cut request list and to open the Plates for Cut Run dialog see Section 8 9 Enter the plate name and barcode plate ID in their respective fields The plate barcode can be entered using a barcode scanner or typed in To reserve wells on a microplate click the Edit button for the desire plate and click on individual wells to reserve them Click Done to complete the cut request list creation 6 2 STARTING A CUT RUN The Analysis Set Excision Control Tool dialog box is used to start a cut run for gels or blots contained in the currently loaded analysis set and to monitor the run progress To start a cut run 1 Make sure the spot cutter is set up as described in Sections 2 and 3 and that the required cutting head is connected to the spot cutter Prepare the gel or blot as described in Section 4 Make sure that the DI water bottle is full and the waste bottle is empty at the start of the run and click Prime Pump If cutting blots fill three 1 5 ml capless microtubes with water to approximately 1 mm below the tube top and then place the tubes into the three membrane tip wash ports A
10. 1 5 mm in the Cut Options panel see Section 8 4 Note that the cut request circle size displayed on the image depends on the tip size selected 9 From the Cut Options panel specify the type of gel to cut see Section 8 4 e Free gels Select this option to use a protocol optimized for cutting non backed gels e Plastic backed gels Select this option to use a protocol optimized for cutting plastic backed gels When this option is used gel plugs are cut and then dislodged from the gel backing by a side to side cutting tip motion e Glass backed gels Select this option to use a protocol optimized for cutting glass backed gels When this option is used gel plugs are cut and then dislodged from the gel backing by a side to side cutting tip motion Note that the cutting tip is raised by 3 mm to accommodate the glass plate e PVDF This option is available only when the spot cutter is in membrane cutting mode and is used to specify that either a PVDF or nitrocellulose membrane will be cut LANE VOLUME EXCISION TOOL Tips for cutting membranes Fill microplate wells with DI water to within 1 to 2 mm of the top In order to release the cut membrane into a microplate well the cutting tip must be in contact with water Do not overfill the wells tubes as this will allow cut membrane to float between adjacent wells 10 Select the vessel type that will be used from the Plates Options panel see Section 8 5 and specify the
11. A steady green light indicates that the spot cutter is powered up and ready for operation A slowly flashing green light indicates that the door is open or the instrument is in operation A rapidly flashing green light indicates the instrument is initializing the robot for operation Pause This flashing yellow light is turned on if the instrument is paused the side panel door is open or the spot cutter is in an error state UV Lamp This blue light indicates that the UV EPI lamps are on Buttons amp Switches Pause Press this button to pause or resume a cut run Light Press this button to turn the Trans white light on or off This button is active only if the door is open Change Cutting Tip Press this button to bring the cutting head to the front of the instrument for changing cutting heads Press it again to return the cutting head to home position This button is active only if the door is open 0 This switch turns the spot cutter power off 0 and on INTRODUCTION 1 2 11 Software The EXQuest spot cutter is controlled through Bio Rad Discovery Series software PDQuest 7 4 0 or greater and Quantity One 4 6 or greater PDQuest Basic and Quantity One Basic software are shipped with the spot cutter and may be activated to the fully featured software by purchase of a separate license agreement PDQuest Basic and Quantity One Basic Software PDQuest Basic and Quantity One Basic software control the EXQuest spot cu
12. Align Analysis and Cutter Images Step 3 Confirm Alignment Panel Click Jump and confirm to visit each spot with an alignment confidence level less than the Confidence threshold If a spot is not correctly aligned click and drag it until it is properly aligned Note that the cursor will display the confidence level as the spot is moved Repeat the above procedure for all spots with a low confidence level Note that the green cut circle size represents the actual cut size and depends on the cutting tip size selected in Step 7 of Section 6 1 ANALYSIS SET EXCISION TOOL Y Align Analysis and Cotter Images sargi cupped v Fitered Daa carers mage ee se eee oH E Spots to be Matched i Ox e ek pp e ma om e i e O Jump an Confirm Spot i Rp o x e p D el D D x E K ol 2 Matched Spots E E f en DD e D SD E A m A TA am Stee 1 Place data bax Siep 2 Parlam abgerert Step 3 Cordero goes Deen R E June ad corie 5 Depley cupped mage JOs net K Cs sen Bs ae Mecie Mito cots Lor ence thesthokt m Corpse cul owedane e SE e o Cute bet theeshokd 2 Tasca 114 Oxe X Cord DI Hep Figure 6 16 Align Analysis and Cutter Images Jump and Confirm The following image options are available in this window Display cropped image Check this box to display only the cutter camera image area outlined by the green data box Uncheck this box to view the entire cutter camera image Cut Marks Check this box to mark
13. FluoeS gt EXQuest Spot Cutter E ChemiDoc EQ tert Mac C ProteomeWorks Spot Cutter 25cm ChemiDoc XRS VersaD gt ess Analysis Set Excision Tool advanced uset mode 465 700 Personal PX 565 710 SI 465 800 Y 0 X Coca E He Figure 3 2 Preferences dialog box 3 Select EXQuest Spot Cutter in the Spot cutter panel EXQUEST CALIBRATION WIZARD 4 Click Setup spot cutter to start the EXQuest Calibration Wizard The screen that will appear depends on the current calibration state of the instrument Spot Cutter Is Not Calibrated If the spot cutter is not currently calibrated the EXQuest Calibration Wizard screen will appear as shown if Figure 3 3 From this screen click Next to focus the camera see Step 5 below i EXQuest Spot Cutter Setup xi EXQuest Calibration Detected EXQuest Spot Cutter with Gel head This Wizard will guide you through calibration You will need the grid template and 9 calibration pucks Place the grid template on the gel tray in the EQuest Spot Cutter as shown dnjas EI o E Click Next to start the focusing dialog Cancel Figure 3 3 EXQuest Calibration Wizard screen uncalibrated Spot Cutter Is Calibrated If the spot cutter is currently calibrated the EXQuest Calibration Wizard screen will appear as shown in Figure 3 4 3 2 EXQUEST CALIBRATION WIZARD x EXQuest Calibration Wizard Detected EXQuest SpotCutter with Ge
14. Gloss AV iii a A aaa a ae tee 12 1 System ee EE 12 2 ii SAFETY SAFETY INFORMATION Read the manual before operating the EXQuest Spot Cutter For technical assistance contact your local Bio Rad representative or in the U S call technical services at 1 800 4BIORAD 1 800 424 6723 CAUTION WARNING e Do not place any part of your body inside the instrument while it is in operation as it could lead to serious injury Do not attempt to override any of the built in safety features and door interlocks Disconnect the power before servicing the spot cutter NOTICE This instrument is intended for laboratory use only This product conforms to the Class A standards for electromagnetic emissions intended for laboratory equipment applications It is possible that emissions from this product may interfere with some sensitive appliances when placed nearby or in the same circuit as those appliances The user should be aware of this potential and take appropriate measures to avoid interference This Bio Rad instrument is designed and certified to meet EN 61010 1 safety standards Operation of this product is subject to the condition that no harmful radio interference is caused and that any interference must be accepted EN 61010 1 certified products are safe to use when operated in accordance with the instruction manual This instrument should not be modified in any way Alteration of this instrument will Void the manufacturer s warranty
15. Me ZG ha e eat E A S fei Al z mm Bas S cds H Je km Vue S D i a L ae pa Ch pa Sin ka e Lo Fues Si aT e M e et Ce m Figure 7 5 The effect of the Horizontal control on the cut requests Left Horizontal 100 Right Horizontal 0 7 3 CUTTING VOLUMES Volume Overlay mode is used to select bands from which cuts will be made 1 Select Volume overlay in the Specify Cuts panel r Specify Cuts Vertical 100 C Entire lane Horizontal 10 Ce Volume overlay El EA X Show excisions on image J Figure 7 6 Lane Volume Excision Tool Specify Cuts panel Volume overlay selected 2 Check the Make multiple cuts on large objects box to make multiple cuts from each band and enter a value in the Maximum cuts object field see Section 8 4 3 Click the Volume Rect Tool and then click and drag a box around the gel bands to cut The box size and the Maximum cuts object value determines the number of cuts per band To adjust the box size hold the cursor over one of the four corners until it changes to a two headed arrow cursor and then click and drag the mouse To move the box place the mouse cursor over the box center until it changes to a four way arrow cursor and then click and drag the cursor Check Show excisions on image to show the cut request labels on the image LANE VOLUME EXCISION TOOL e mg n e dia ote M
16. The lightest image pixels will be set to the minimum intensity white and the darkest pixels will be set to the maximum intensity black This maximizes the display range for the image You can then fine tune the display using the sliders as described above Image Color Click this button to open a color map list Color maps can be defined using the Colors command on the Edit menu Select a color map from the list to change the image color in both the preview and image window Reset Click Reset to cancel all transform changes and return to an unmodified view of the image data OK Click OK to apply the transform settings to the image and exit the Transform dialog e Cancel Click Cancel to exit the Transform dialog without applying the image transform 8 4 CUT OPTIONS PANEL Specify Options Cut Plate Hydration Wash X Make multiple cuts when possible Maximum cuts object 3 Cutting tip size 1 0mm 1 5 mm Material Free gel Figure 8 4 Cut Options panel The Cut Options panel is used to select the cutting tip size the gel blot type and the cut parameters It may appear in a tab box as shown above or as a separate panel Make multiple cuts on large objects Check this box to make multiple cuts from large spots Check this option to increase protein yields Maximum cuts object This field is used to set the maximum cuts that can be made from a single spot The number of cuts made from lar
17. UPS recommended Computer Minimum Requirements e Processor 1 2 GHz or greater e USB 1 1 or 2 0 e 2 1 GB hard drive RAM 512 MB or greater e Screen Resolution 1 024 x 768 pixels e 2 MB video RAM e Operating system Windows 2000 or Windows XP Environment e Operating Temperature 15 30 C e Humidity 15 85 non condensing 2 2 UNPACKING THE EXQUEST SPOT CUTTER The EXQuest spot cutter and its accessories are shipped together in a single palleted crate Before unpacking the EXQuest spot cutter carefully read the unpacking instructions located on the outside of the packaging Inspect the container for any damage that may have occurred in shipping Severe damage to a container may indicate damage to its contents If you suspect damage may have occurred immediately file a claim with the carrier in accordance with their instructions before contacting Bio Rad Laboratories SYSTEM INSTALLATION A Caution The EXQuest spot cutter must be lifted by two or more individuals from its shipping pallet using the removable lifting handles attached to the spot cutter Do not lift the instrument from the lower front bezel The spot cutter must remain in an upright position at all times and be placed on a sturdy level surface The EXQuest spot cutter is shipped with the following components EXQuest spot cutter e Camera CD containing PDQuest and Quantity One Basic software in separate package Power cord e USB cable Bottles for Dl water
18. aa adas 2 3 2 4 Gel Tray Installation cui aa 2 3 2 5 Microplate Rack Installation oo nnoccninninicinnnnnncoccnnnncccccnnnnncnancn nn nnnn cnn aran r anna 2 4 2 6 Cable SOU iinis eri A ate dE EN 2 5 2 7 Cutting Head Installation 00 ecccececeeeeeeeeeeeeeeeeeee eee aeeeseaeeseeaeeeeeaaeseeaeeesaaeeseaeeeseeeeeeaeesseas 2 6 SCT Gel Cutting Head dt 2 6 2 7 2 Membrane Cutting Head 2 7 2 8 DI Water and Waste Bottle Installation oon nniiininiinnn nncccnnnnnacccncnnnncccrnnrrnnnarnn rra nc 2 8 2 9 Software Installation AAA 2 8 2 10 Starting the Software iia A diel ete 2 9 SECTION 3 EXQUEST CALIBRATION WIZARD ccccesceseseeeeeeeeeseeeeeeeeeeeseaaeenseeeeeenessaseeeeeseeeeeneaeeenenees 3 1 SECTION A GEL AND BLOT PREPARATION scseccseseeeseeeeeeseeeesseeeneeeeessaaeenseeeeseesseneeeeeseeeseseaeeeeenees 4 1 4al Free ET 4 1 4 2 Plastic and Glass Backed Gels conmccnncccninncnnnocncconcccononcnnnnn cnn rca n anna 4 2 4 3 Blot PreparatliOn EE ad ipdca 4 2 SECTION 5 MANUAL EXCISION TOOL ooncmccconncncnicccninncrcnnir rr 5 1 5 1 Performing a Manual Excision Tool Cut RUN oonncccnnccconnccnnoncccnnnnncnnno cocoa rra nn 5 1 Elon indie E AT 5 4 5 3 Confirm Cuisine 5 5 5 3 1 Identifying Re cuts From a Microplate Image oooonninnccnnnnnnccccnnnncccccnnnnanrcnnnn anni 5 5 5 3 2 Identifying Re cuts From a Gel Image 5 7 SECTION 6 ANALYSIS SET EXCISION TOOL ommcncicccnnnnnonincccnnncarenrer nc 6 1 6 1 Setting Up An Analysis Set
19. and well assignment SSP Displays the Standard SPot number Gel The Analysis Set image file name Quantity Displays the spot intensity Cuts Displays the number of cuts that will be made for a single spot Cut date Displays the date that the spot was cut Status Indicates whether the spot has been cut or not Check Hydrate to activate automatic gel hydration and then specify how often the gel is to be hydrated see Section 8 6 If hydration will not be performed uncheck the Hydrate box Gels and membranes should typically be hydrated at least once every hour If the gel head is installed enter the water volume required to wash the gel cutting tip 50 ul to 500 ul in the Wash Options panel see Section 8 7 If a blot is to be cut fill three 1 5 ml microtubes with water to approximately 1 to 2 mm below the top and place the tubes in the membrane tip wash ports Press Go to open the Place Gel Blot on Spot Cutter dialog see Figure 6 10 and 6 11 ANALYSIS SET EXCISION TOOL 10 11 12 13 6 8 id Place GeVBilet on Spot Cutter Step 1 Place gail sl on cotter Diren ge gei onthe cutter the conte ofthe stage When the gelis on the cutter press Acquare image Kaes Fe Tiara Pro nera Tee EEE o Duplay Arayin mapas bom gel placement Figure 6 10 Place Gel Blot on Spot Cutter dialog box Single Gel Placement Place Gels Blots on Spot Cutter Step 1 Place gel s on cutter Step 2 Acquire image of gel
20. as part of the run The ml well volume must be greater than or equal to Min Well Volume Loading Order Select A1 A2 for a front to back fill order or A1 B1 for a left to right fill order 8 6 HYDRATION OPTIONS PANEL Specify Options Cut Plate Hydration Wash x Hydrate Every 60 0 minutes Hydrate now El Define hydration area Figure 8 6 Hydration Options panel The Hydrate Options panel is used to set up hydration parameters Hydrate Check this box to turn auto hydration on and to activate the hydration interval field Enter the hydration interval in minutes The interval time should usually be set to approximately one hour The actual time required will depend on your laboratory temperature and humidity EXCISION TOOL CONTROLS Define hydration area To define the cutting deck area to hydrate click this button and then click and drag a box around the gel or blot This function is not present in the Analysis Set Excision Tool since the gel location is identified by the position of the green data area box in the Align Analysis and Cutter Images window Hydrate Now Click this button to immediately hydrate the gel Note that if Hydrate is checked the gel blot will be hydrated before the run starts 8 7 WASH OPTIONS PANEL Specify Options Cut Plate Hydration Wash Wash volume 50 0 ul Figure 8 7 Wash Options panel The Wash Options panel is used to
21. axis is responsible for cutting gels and membranes Caution do not place body parts or other objects inside the EXQuest spot cutter while it is in operation as this may cause serious injury and damage to the instrument Figure 1 9 Robotics 1 7 INTRODUCTION 1 2 9 Sensors The spot cutter includes a series of sensors to protect the operator and ensure the proper operation of the instrument Front Door Sensor When this sensor detects that the front door is open it turns off the UV EPI fluorescent lights causes the spot cutter to operate at reduced speed and prevents images from being taken Water Waste Bottle Door Sensor When this sensor detects that the bottle door is open it pauses the instrument UV Light Sensor This sensor determines if the UV lights are on and ready before acquiring an image Waste Reservoir Level Sensor This sensor activates the waste pump when the wash port reservoir is full DI Water Sensor This sensor is used to warn the operator that the water level is low Waste Sensor This sensor is used to warn the operator that the waste bottle is full Tray Sensor This sensor is used to warn the operator that the gel tray is not installed A cut run cannot be started if the tray is not fully inserted 1 2 10 Front Panel Controls and Indicators Change i Pause Light Cutting Tip EI E O 9 UV Lamp Figure 1 10 Status light and front panel controls Indicators Status
22. filled with water to within 2 to 3 mm of the top Gels that are not properly hydrated may shrink or swell If gels are becoming dry increase the hydration rate Fully hydrate gels before setting up a cut run Gels or membranes that are overhydrated may float This is particularly problematic for membranes and backed gels Make sure clamps are used to hold the gels and membranes in place and do not over hydrate Before placing backed gels on the cutting sheet dry both the cutting sheet and the gel backing Recalibrate the spot cutter Repeat the calibration Examine calibration pucks for material blocking the hole at the bottom of the V shaped well Examine the pucks for damage that does not allow the puck to center properly Replace damaged pucks Restart the calibration procedure 11 2 TROUBLESHOOTING If all the settings seem to be correct contact Bio Rad for further troubleshooting For technical assistance with the system including all hardware and software contact your local Bio Rad office All spare parts not listed in this document can be ordered by contacting your local Bio Rad office For inquiries and requests with the system including system repair or service contact your local Bio Rad office or distributor Please have the following details available 1 Instrument model and catalog number 2 Serial number located on the back of the system and on the inside of the bottle access door 3 Hardware and so
23. makes no warranty whatsoever in regard to products or parts furnished by third parties such being subject to the warranty of their respective manufacturers Service under this warranty shall be requested by contacting your nearest Bio Rad office This warranty does not extend to any instruments or parts thereof that have been subject to misuse neglect or accident or that have been modified or serviced by anyone other than Bio Rad or its representative or that have been used in violation of Bio Rad instructions It also does not extend to instruments or parts thereof that have been used with fittings or other spare parts not authorized by Bio Rad Laboratories that are interfaced to inappropriate external devices that have been exposed to inappropriate solvents cleaning agents or samples The warranty also does not cover instrument damage resulting from facility problems such as power surges The foregoing obligations are in lieu of all other obligations and liabilities including negligence and all warranties of merchantability fitness for a particular purpose otherwise expressed or implied in fact or by law and state Bio Rad s entire and exclusive liability and buyers exclusive remedy for any claims or damages in connection with the furnishing of goods or parts their design suitability for use installation and operation Bio Rad Laboratories will in no event be liable for any special incidental or consequential damages whatsoever and Bio Rad s li
24. need to be filled with water or an appropriate wash solution to within 1 to 2 mm of the tube top before they are placed in the membrane tip wash ports During a cut run the tip is dipped in one of the three tubes after each cut to both rinse the membrane tip s outer surface and ensure that any attached membrane debris is removed from the cutting tip Microplate Rack Widely 7 Closely spaced tabs spaced tabs N Figure 1 7 Microplate rack The microplate rack is a two position reversable rack located to the left of the gel tray It holds microplates that have a foot print compatible with the SBS standard for microplates When the rack is positioned as shown in the figure above it accommodates standard height microplates 14 mm high When inverted it accommodates deep well microplates 41 to 44 mm high and microtube racks 41 to 48 mm high The microplate rack holds up to four 96 tube racks 96 well plates or 384 well plates 1 6 INTRODUCTION Figure 1 8 Supported microplate formats The microplate rack accommodates plates from a variety of vendors that include Corning Whatman Greiner AxyGen and Bio Rad Microplates with a U or V bottom are recommended for best sample recovery and are available from Bio Rad see Accessories Section 9 1 1 2 8 Robotics The EXQuest robot is a precision device that moves the cutting tip between the gel cutting area microplate rack and wash station Motion of the vertical
25. of the pucks move the pucks to the correct position refine the puck placement take another image and then finish the calibration Alternatively click Back to repeat the lens calibration or Cancel to abort the calibration Remove the protective sleeve if the membrane cutting head was calibrated Remove the calibration pucks and store them in the box provided to prevent them from being damaged Once the System Position Calibration has been completed it will automatically save the calibration You may now exit Cancel the calibration wizard without losing the calibration 10 Plate Position Calibration The plate position calibration step is used to center the cutting tip over the microplate wells a Place a microplate with the desired format in the back right slot of the microplate rack with well A1 located in the front left corner of the microplate Select the microplate format and then click Next 3 7 EXQUEST CALIBRATION WIZARD EXQuest Spot Cutter Setup xj Plate Position Calibration Remove the calibration pucks from the stage Select the type of container you would like to calibrate 96 well Microbter Plate C Ziel Microtter Plate C S6 tube Rack Dick Nest to continue dnjas senox3 Figure 3 10 Plate position calibration plate selection b Use the Left Right and Front Back slider to center the cutting tip over well A1 as shown below in Figure 3 11 Click on the slider bar to move the tip
26. position PO Quest wil move the cutting head and wait for you to postion each puck under the head in sequence dnjas ECE o E Cick Next when ready lt Bock Next gt Cancel Figure 3 9 System position calibration Manual Calibration a f g If calibrating the membrane cutting head install the plastic protective sleeve over the 1 0 mm cutting tip Click Next to start the system position calibration and move the cutting head to the first position or click Back to repeat the lens calibration step or Cancel to abort the calibration Center a puck under the cutting tip and then click Next to position the puck and move the cutting head over the next puck Back to reposition the previous puck or Cancel to abort the calibration Continue as described in Step b until all the pucks have been positioned Click Done and close the front door The spot cutter will refine the puck placement acquire an image of the pucks and then finish the calibration Remove the protective sleeve if the membrane cutting head was calibrated Remove the calibration pucks and store them in the box provided to prevent them from being damaged Automatic Calibration a d e If calibrating the membrane cutting head install the plastic protective sleeve over the 1 0 mm cutting tip Check the Automatically move pucks to alignment position box Click Next to perform an automatic puck placement The spot cutter will take an image
27. repeat steps 8 through 16 18 Optional After a cut run is complete you may re cut spots on the gel if desired This is generally not necessary due to the high cutting efficiency of the EXQuest spot cutter To perform re cuts a Click Yes in the Acquire confirmation image dialog box that appears at the end of the run A confirmation image will be acquired Use the main tool bar zoom function to zoom in on the individual cuts When a missed spot is identified click in the Analysis Set Control Tool and check the box corresponding to the missed spot d When all the missed spots have been identified click Ready to re cut the spots or click Cancel if no missed spots were observed 6 3 ANALYSIS SET EXCISION TOOL ADVANCED USER MODE The PDQuest Advanced User Mode is used to automatically generate cut request lists see Automatic Gel Selection in Section 6 1 Step 5 which can then be edited manually to add delete or pool cuts To operate PDQuest software in Advanced User Mode 1 Start the PDQuest software and click Edit Preferences File ES View Image Spots Text Overlays gt ca Calibrate Gels gt Preferences Colors Mouse Mouse Macros b Figure 6 19 Edit Pull down Menu 2 Click on the Devices tab in the Preferences dialog D Preferences Mec Paths imagers Select the Imagers to be diplayed in the fle menu x Gel Doc EQ x FluorS X Gel Doc XA 5 F
28. spots in the analysis set image with blue X s These marks cannot be seen if Matches is checked Matches Check this box to mark matched spots with green and red circles in both the analysis set image and cutter camera image Auto zoom Check this box to cause the camera cutter image right side to mimic the zoom state of the analysis set image left side Cut Area Check this box to overlay the green cutting area boundary box on the cutter image The cutting head cannot cut spots outside this area Multiple Cuts Check this box to display Multiple cut spots Data box Check this box to display the green data area box Uncheck this box H the data area box is obstructing the view of spots on the periphery Compute cut overlaps The Compute cut overlaps function automatically calculates the percentage that spots are overlapped Cut Overlaps Check this box to mark overlapping spots with ellipses in the analysis set image The percent overlap will be displayed next to the overlapped cluster In the cutter image click and drag overlapped spots to adjust their position 6 11 ANALYSIS SET EXCISION TOOL sample v1 Fi em SEN 85 es ae 5 gt iy RK 5 775 o Figure 6 17 Align Analysis and Cutter Images Cut Overlaps 15 Click Proceed to hydrate the gel or blot if hydration is turned on and open the Load Plates on Cutter dialog box see Section 8 10 Locate the microplate with the name and barcode plate ID shown in th
29. tray and align it with the front left corner of the gel tray Click Next to perform the lens calibration Back to repeat the previous step or Cancel to abort the calibration G EXQuest Spot Cutter Setup E xj Lens Calibration This step requires the Can aber template gid suppbed with this system Make sure the grid is clean dry and flat Place the gud on the gel tray and pul A toward the front left comer so that R it aligned with the tray sides as shown below When the gid it properly aligned close the door and click Next dnjas sanoxX3 Figure 3 8 Lens Calibration screen System Position Calibration The system position calibration step aligns the image coordinates with those of the robot Remove the calibration target from the gel tray and place 9 calibration pucks in a 3 x 3 evenly spaced grid on the gel tray as shown in Figure 3 9 Perform an automatic or manual calibration Note that automatic and manual calibration will give identical results However automatic calibration requires no user interaction Automatic calibration is only available if the spot cutter has been previously calibrated EXQUEST CALIBRATION WIZARD D EXQuest Spot Cutter Setup xj System Position Calibration Remove the grid template from the gel tray Place the calibration pucks in a 3x3 evendp spaced grid on the gel tray making sure the well tide is up as thown FN O JAutomabcaly move pucks to exact alignenent
30. 1 0 mm Nominal Gel Cutting Tip R 165 7203 EXQuest 1 5 mm Nominal Gel Cutting Tip R 165 7206 EXQuest 1 0 mm Nominal Membrane Cutting Tip A 165 7204 EXQuest DI Water or Waste bottles R 165 7210 EXQuest Calibration Target Template R 165 7215 Gel Tray R 170 3751 UV Lamps 4 required R 165 7206 EXQuest Membrane Head A 165 7218 EXQuest Membrane Head Tip Protector R pkg of 10 165 7212 1 5 ml Capless Microtubes R 165 7219 Barcode Reader A 165 7217 96 well microtiter plate pkg of 20 MAINTENANCE 10 0 MAINTENANCE 10 1 ROUTINE MAINTENANCE e The gel tray should be cleaned after use with a damp nonabrasive cloth Mild detergents may be used Do not use any harsh solvents or cleaners e The gel tip should be inspected prior to use Both the gel and membrane cutting tips are made of high quality 303 304 316 stainless steel If properly maintained these cutting tips will retain their cutting ability through repeated use Replacement is needed if the cutting tips have been bent or damaged in any way If the cutting tip has jammed against any solid object it should be inspected and replaced if necessary After each use the gel tip should be washed with methanol or ethanol e At least monthly inspect the wash station filter for the presence of gel plugs and clean it if necessary The wash station filter is cleaned by unlatching the coiled tubing open it by rotating the two halfs in counter clockwise direction and sepa
31. Excision RUN c ccccecceseceeeeeeeeeceeeeeeeeeeeeaeeeseaaeeseeeeetaeenennees 6 1 Ginet her el TEE 6 6 6 3 Analysis Set Excision Tool Advanced User Mode oocooiccccncconocicocncconccnoncnnn con nanancnnnannnann 6 13 TABLE OF CONTENTS SECTION 7 LANE VOLUME EXCISION TOOL cscccesseeseeeeeeeeeeeseeeeeneeeeessaeseeseeeeeeeeseaneeeenseeeeaneaeeneeeees 7 1 7A Setting Up a C t R N cai edie eile healt a a aa 7 1 Ke ug e NEIE 7 2 1S Gutting el 7 4 SECTION 8 EXCISION TOOL CONTROLS cccescccsceeeeeeteeesneeeeneeeeesnaeeeneeeeeseaaesesneeeseaesseneeeeeseeesneaeeenseees 8 1 8 1 ee CH ue CHE 8 1 8 2 Image Controls viciosa da fa 8 1 8 3 Transtorm Dialog E 8 2 8 4 Guttptnens Panel AA AD E td vara 8 3 8 5 Plate Options Panel ricino siria a pd 8 4 8 6 Hydration Options Panel 8 4 8 1 Wash Options Pelis cia a E 8 5 8 8 Run into Panel iii dida 8 5 8 9 Plates for C t RUN Lata ta a a 8 6 8 10 Load Plate on Cutter Dialog 8 7 SECTION S ACCESSORIES ovocitos 9 1 SECTION 10 MAINTENANCE oomcococccconcncicrncnnnnnr rn 10 1 10 1 Routine Maintenance conmcccccnnoncccccnnnoneccnnnnnnecnnonnnneconn nan EA E AE E AA T E E 10 1 10 2 UV Light Replacement is cerono tiisa ii e aeaoe aeii ie ida 10 1 1 0 3 FUSES oonu E e A a a a a raras 10 2 SECTION 11 TROUBLESHOOTING aaa raar raea reaa anana re dapa aai aoe Soni anarai esain ie raaa aKo attu iek oda kadindan 11 1 WARRANTY aa E EE E E EE E T ti vetvasccdestbecneedtaeene 12 1
32. I lamp end cap toward the front of the spot cutter to remove the lamp assembly 10 1 MAINTENANCE Figure 10 2 UV EPI Lamp Partially Removed 3 Place the lamp assembly upside down on a flat surface and then rotate each fluorescent tube 90 degrees Once the metal pins are visible lift the tubes out and dispose of them Figure 10 3 UV EPI Lamp Replacing the Fluorescent Tubes 4 Reverse the procedure to install the four new fluorescent tubes 10 3 FUSES To replace a fuse Turn the power switch off and unplug the spot cutter from its power source Squeeze the fuse door tabs and pull it open see Figure 10 4 Remove the old fuse and then insert a new Type T 2 5A fuse Replace the fuse door reconnect the power and turn the instrument on PON gt Note If the instrument does not power up make sure the fuse was installed correctly In the event the new fuse has blown or the instrument will not power up contact your Bio Rad technical representative immediately 10 2 MAINTENANCE Fuses Type T 2 5A POWER ENTRY Figure 10 4 Spot cutter fuse 10 3 11 0 TROUBLESHOOTING TROUBLESHOOTING Symptom Cause Solution Computer does not USB cable not Ensure that both the USB cable and power cord communicate with connected are connected spot cutter or camera Poor image quality Low gel cutting efficiency Computer went into sleep mode The spot cutter power switch has not been turned on Camera or
33. Read Me Screen 9 Enter your Full Name and Organization and then click Next 10 Select the installation directory and click Next 11 Click Next and follow the rest of the on screen instructions to complete the installation 12 When the installation is complete shut down the computer to complete power off and then power it back on Make sure the EXQuest spot cutter has been powered on and fully initialized before starting the PDQuest or Quantity One software 2 10 STARTING THE SOFTWARE 1 Double click the PDQuest or Quantity One desktop icon to start the software Alternatively from the Start menu open the All Programs The Discovery Series folder and select PDQuest or Quantity One Software License 9 Welcome to PDQuest Current License Basic i Enter Password Unable to Register Bee The system ID can not be determined for this computer Registration Form and is required for software registration If you have a Hardware Protection Key please install it or check to make sure it is installed properly See Release Notes Exit PDQuest Otherwise contact your Bio Rad representative to order a Hardware Protection Key Help Basic mode Allows you to acquire new images open existing images perform basic editing functions print export to TIFF and Save Click Help for further information Contact your local Bio Rad office for information on purchasing a full user license or visit us on line at www bio rad com
34. Set Excision Tool Manual Excision Tool E WD Mass Spec Score Overlay Figure 6 1 Identify pull down menu 3 Click the Name down arrow and select the desired analysis set If a description of the analysis set has been entered previously it will be displayed in the Description field The number of spots contained in the analysis set will be displayed in the Spot count field Analysis Set Excision Wizard Analysis Set Excision Wizard This Wizard will guide you through setting up an excision run Select the analysis set that contains the spots you want to excise Name Sample Analysis Set D Descaption Spot count 204 UOISIDXF 18S sishjeuy Next gt Figure 6 2 Wizard Analysis Set Selection ANALYSIS SET EXCISION TOOL 4 Click Next to continue The wizard screen that appears will depend on whether a cut request list has already been created or not These screens will allow you to e Create a cut request list T Analysis Set Excision Wizard Gel Selection Method You may select gels to cut either manually or automatically Maul gel selecton C Automatic gel selection UOISIDXF 19S sisAjeuy Figure 6 3 Gel Selection Method From the Gel Selection Method screen Figure 6 3 Select Manual gel selection and then click Next to manually choose one or more gels to cut Go to Step 5 Manual gel selection Select Automatic gel selection and then click Next to allow the software
35. ability under no circumstances will exceed the contract price for the goods for which liability is claimed GLOSSARY Click Unless otherwise specified this refers to a single left button mouse click on the desired menu or item Panel A panel is a segment inside a window or screen containing related controls and information Press Unless otherwise specified this refers to a single left button mouse click on the desired menu or item Select Unless otherwise specified this refers to a single left button mouse click on the desired menu or item Spot A spot is an image position that is selected to be cut Screen This generally refers to a dialog box contained in a wizard Text Field Is an area into which text can be entered Window Is either i Similar to a dialog box except it contains text fields and tabs which can be manipulated or ii A view window which contains a picture 12 1 WARRANTY SYSTEM RECORDS Serial Number Date of Purchase Purchase Order Number Notes Service Record Date Description of Problem Name Signature 12 2 Life Science Group Bio Rad Laboratories Inc Web site www bio rad com USA 800 4BIORAD Australia 02 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 21 2527 3454 Canada 905 712 2771 China 86 21 6426 0808 Czech Republic 420 2 41 43 05 32 Denmark 44 52 10 00 Fin
36. accepted the cut requests you may move individual cuts using the Add move cut function Continue setting up the cut run as described in Section 5 1 Step 14 5 3 CONFIRM CUTS The Confirm cuts function is used to identify missed gel spots and is particularly useful for fluorescent stained gels since the clear gel spots with no visible stain are difficult to see in microplate wells The confirm cuts function can be used to image microplates to locate wells that do not have gel plugs in them or to image gels to locate gel plugs that have not been picked up Its use is optional and generally not required due to the EXQuest spot cutter s high cutting efficiency 5 3 1 1 Identifying Re cuts from a Microplate Image The Confirm cuts pop up box automatically appears at the end of a cut run see Figure 5 6 below when the gel has been imaged with the UV EPI light Click Yes to acquire an image of the microplates or No to return to the Manual Excision Tool window MANUAL EXCISION TOOL 2 10 11 il PDQuest Confirm cuts Acquire an image of the MTP in U mode to check whether re cuts are needed y Yes X No Figure 5 6 Confirm cuis pop up Read the instructions on the Image Microtiter Plate screen O Image Microtites Plate Step 1 Setup MTP A not move gel on stage Cover gel h Gel Cutting Steet Place microtiter plate on Gel Cutting Sheet Glove door and cick Acqua Image LE Ste
37. ailed description of each component is given below Robot UV EPI lights Water and Microplate Wash Gel Tray and Controls and Waste Bottles Tray Station Transilluminator Status Lights Figure 1 2 EXQuest Spot Cutter 1 2 1 Enclosure The EXQuest spot cutter enclosure provides protection from ambient dust and contamination and acts as a dark enclosure for fluorescent imaging To open the front door raise the door to the top and then lower it until it latches To close the door lift the door and then lower it until it is closed 1 2 2 INTRODUCTION Figure 1 3 Light enclosure Imaging System The gel and blot imaging system consists of a CCD camera 1600 x 1200 pixels 12 bit ADC pixel size 230 microns fitted with a 620 Center line 60 width nm band pass filter that is optimized for SYPRO Ruby protein gel stain It also encompasses two fluorescent UV EPI lights located above the gel tray and a Trans white light source located under the gel tray 1 23 Gel Tray and Gel Clamps The gel tray securely holds gels and membranes to be imaged and cut Its features include Large surface area The gel tray s large size 32 x 28 cm allows it to hold one large gel or up to four small gels e g Criterion gels without the need for trimming Gel placement guides The gel tray is marked to help position gel s and blots Gels and blots should be positioned near the center of the gel tray for best performance Removab
38. amps on the blot Insert the gel tray into the spot cutter and make sure it is latched securely by pushing it toward the rear until it snaps into place MANUAL EXCISION TOOL 5 0 MANUAL EXCISION TOOL The Manual Excision Tool is a general purpose excision tool included in both the PDQuest and Quantity One software It has the basic functionality required to image gels make cut requests and cut gels See the PDQuest and Quantity One manuals for more information 5 1 PERFORMING A MANUAL EXCISION TOOL CUT RUN 1 Make sure the spot cutter is set up as described in Sections 2 and 3 and that the required cutting head and tip is connected to the spot cutter 2 Prepare the gel or blot as described in Section 4 3 Open the PDQuest software and click Identify Manual Excision Tool or open the Quantity One software and click Excision Manual Excision Tool see Figure 5 1 e Identify Report Window Help Analysis Set Excision Tool 2 xi Manual Excision Tool Mass Spec Score Overlay Figure 5 1 Opening the Manual Excision Tool in PDQuest and Quantity One 4 Make sure that the DI water bottle is full and the waste bottle is empty before you start and click Prime Pump 5 When cutting blots fill three 1 5 ml capless microtubes with water to approximately 1 mm below the tube top and place them in the three membrane tip wash wells see Section 1 2 6 After making a cut the cutting tip will be dipped into o
39. and waste 2 Bottle holder Gel cutting heads 1 0 mm and 1 5 mm Gel cutting sheets 15 Microplate rack Gel tray Gel Blot clamps 4 Calibration target Calibration pucks 10 9 64 Hex wrench User manual Packing list Unpacking instructions An installation kit comprised of one fittings wrench wrench for leveling feet bubble level four blotting paper sheets and four 96 well plates The following components will be shipped separately if ordered Computer Shipped in two boxes that will contain Box 1 Computer keyboard mouse power cords manual and system disks and Box 2 17 flat screen color monitor PVDF and nitrocellulose membrane cutting head Includes Membrane cutting head Membrane cutting tip 1 0 mm 1 Caution tips are sharp e Cutting tip installation wrench 2 e Capless microtubes 1 pkg Packing list If any parts are missing or damaged contact Bio Rad Laboratories immediately SYSTEM INSTALLATION 2 3 SYSTEM LEVELING To level the EXQuest spot cutter 1 Place the spot cutter on a lab bench that is sturdy and level 2 Loosen the 9 16 inch lock nuts on the spot cutter legs and remove the lifting brackets The handles on each end of the lifting bracket are removable so that the lifting bracket can be slid out from under the instrument 3 Place the bubble level that was shipped with the instrument on the base plate immediately to the left of th
40. be placed about 2 mm into its wash port and the Membrane cutting tip is about 2 mm or more into its wash port Note that the membrane cutting tip must be in contact with the water in the microtube in order to effectively clean the tip d Click Next to save the wash port calibration Back to return to the previous step or Cancel to abort the calibration 12 Calibration Complete The calibration complete step displays the Calibration Summary for the calibration steps performed Click Finish to exit the Calibration Wizard 3 10 EXQUEST CALIBRATION WIZARD dnjas senz View log Finish Figure 3 14 Calibration complete screen 3 11 GEL AND BLOT PREPARATION 4 0 GEL AND BLOT PREPARATION Gel and blot preparation is an important part of setting up a successful cut run Failure to prepare gels and blots properly may result in loss of accuracy and or decreased cutting efficiency Gel and blot preparation is described in Section 4 1 free gels Section 4 2 plastic and glass backed gels and Section 4 2 blots 4 1 FREE GELS 1 Store gels in a container that keeps them protected and hydrated until needed for spot cutting Before a gel is cut it is best to equilibrate it in water for at least 1 2 hours or preferably overnight and then to bring it to room temperature This will greatly reduce any swelling that may occur when the gel is hydrated on the gel tray 2 Remove the gel tray from the spot cutter place a cutting s
41. cked gel to prevent the backing from sliding during the cut run 2 Remove the gel tray from the spot cutter then transfer the backed gel onto it 3 Ifa single gel is being cut position it in the middle of the gel tray see Figure 4 1A If two to four small gels are being cut using the Analysis Set Excision Tool position them as indicated on the screen see Figure 4 1B Avoid excess water on the cutting sheet as this may cause the gel to float and decrease the spot cutting performance Position the gel clamps on or against the gel or gel backing Insert the gel tray into the spot cutter and make sure it is latched securely see Section 2 4 hd GEL AND BLOT PREPARATION 4 3 BLOT PREPARATION 1 4 2 Store PVDF and nitrocellulose blots in a container that keeps them protected and hydrated until needed for spot cutting Remove the gel tray from the spot cutter and place a cutting sheet on it A cutting sheet should always be used to cover and protect the gel tray from being scored by the cutting tip Remove the thin plastic film from the cutting sheet before using it If a single blot is being cut position it in the middle of the gel tray see Figure 4 1A If two to four small blots are being cut using the Analysis Set Excision Tool position them as indicated on the screen see Figure 4 1B Avoid excess water on the cutting sheet as this may cause the blot to float and decrease the spot cutting performance Position the gel cl
42. cutting sheet as it is important for cutting efficiency If the spot cutter is not cutting efficiently recalibrate the tip height as described in Section 3 Gels that become dry are not picked up efficiently If gels become dry increase the hydration frequency TROUBLESHOOTING Symptom Cause Solution Low membrane cutting efficiency Low membrane delivery efficiency Low gel and membrane cutting accuracy Calibration fails Hydration and clamping Tip is dull or damaged Microplate wells and tubes need water Gel hydration Hydration and clamping The spot cutter is out of calibration Calibration puck is misaligned Calibration pucks are damaged An error occurred during the flat field or lens calibration Excess water on the gel tray causes backed gels to slide when the gel plug is being scraped off the backing Make sure clamps are used to hold gels in place and do not over hydrate Before placing backed gels on the cutting sheet dry both the cutting sheet and gel backing A membrane cutting tip that is dull or damaged will not cut PVDF and nitrocellulose membranes efficiently If the tip is dull or damaged replace the tip The membrane cutting tip must contact water to efficiently release cut PVDF and nitrocellulose membrane Fill microplate wells within 1 to 2 mm of the top and wash station microtubes to within 3 to 4 mm of the top Make sure the three membrane wash station tubes are
43. e Gamma r Y OK Japan Autos X Cancel DI Help Autoscale Reset lI mage color Figure 8 3 Transform Dialog Transform Controls 8 2 Full scale Low High Select the Full scale option to display the full frequency distribution or the Low High option to magnify the frequency distribution between the High and Low sliders Log Check the Log checkbox to change the way data are displayed in the histogram This function is used to better discern subtle changes in signal intensity High Low sliders Use the High and Low sliders to optimize the preview image manually Drag the High slider handle to the left to make weak signals appear darker Drag the Low slider handle to the right to reduce preview image background As you drag the sliders examine the Frequency Distribution Histogram Everything to the left of the Low slider will be remapped to minimum intensity while everything to the right of the High slider will be remapped to maximum intensity Position the markers at the ends of the data range in the histogram and use the low slider to cut off the spike of background noise You can also type specific high and low values in the fields to the right of each slider Click anywhere on the slider bars to move them incrementally If the image data are in a narrow range select the Log High Low Sliders check box This allows finer adjustments with the slider handles Gamma Slider Some images may be more effectively
44. e software will calculate the number of cuts to make based on the box size If you want multiple cuts from a single spot to be placed in different microplate wells deselect the Make multiple cuts on large objects option in the Cut options tab uncheck Snap to peak and then click on the spot multiple times to make multiple cut requests When cut requests are made they are given a spot number that is later changed to a microplate well assignment when Begin Resume is clicked see step 17 below Each selected cut request is depicted as a circle with a diameter that represents the cutting tip size 1 0 mm or 1 5 mm To annotate an image with the cut request numbers microplate well assignments check the Show Well box To annotate the image with the spot X and Y coordinates check the Show Position mm box Show JPatell Positior rom Figure 5 4 Show well and position checkboxes 15 Review the cut requests by zooming in on them see Section 8 2 and then examining them closely e To move a cut click Add move cut place the Add Move Cut cursor over the cut it will change to a 4 way arrow then click and drag the cut circle to its new location To delete a cut request click Remove cut and then click on the cut circle or center cut circle of a multiple cut box to delete it To remove several cut requests at a time click and drag a box around all the cuts to be removed 16 Optional review the Run Info panel see Section 8 8
45. e dialog and then scan or type in the barcode plate ID into the Confirm barcode field If the Confirm barcode does not match the Barcode a warning will pop up that states there is a barcode mismatch In this case make sure that you have the correct microplate and have entered the barcode correctly Place the microplate in the microplate rack position identified by the green box with well A1 located in the front left plate corner Click Done to proceed to the next plate or Prev to return to the previous plate Click Finish to start the run 16 Optional Monitor the run progress in the Analysis Set Excision Control Tool As the run progresses each cut is displayed in the Member image and Cutter image panels In addition the cut status is changed to In progress and then to Completed in the Cut List table U Analysis Set Excision Control Tool Cit kat Analyse set name Sampie Anatysis Set Gel spots to cut 52 Anahyus vet spot count 204 Total cuts inci multi 2 Cul MTP 3812 3 01 3 02 3 003 3 004 x 3006 x 3 006 si ACHT 3 08 Si 3 09 Bis Cut g e KS 28 Jan 20 20 Jan 20 28 Jan 20 26 Jan 20 Wet 3 COS Spot 17 of 52 OUt 1 of 1 Q Pane Figure 6 18 RENE Set Excision Control Tool Run In Progress 6 12 ANALYSIS SET EXCISION TOOL 17 When the run is finished you will be prompted to place the next gel on the spot cutter if you are cutting multiple gels Place the gel on the gel tray and
46. e gel tray The bubble level should be placed as close to the center as possible Use a 5 8 inch wrench to adjust each leg until the spot cutter is leveled 5 Once the instrument is leveled tighten the lock nuts with the 9 16 inch wrench Lock Nut 5 8 Leg Adjustment E Figure 2 1 Spot cutter leg 2 4 GEL TRAY INSTALLATION Open the spot cutter front door and insert the tray deep side up in the tray guides that are located above the light box as shown below To insert the tray push it in until it latches To remove the tray pull it straight out Note that each latch must be pointing outward before the gel tray can be inserted Tray ie latches Tray Guides _ Figure 2 2 Gel tray installation SYSTEM INSTALLATION 2 5 MICROPLATE RACK INSTALLATION The microplate rack is installed to the left of the gel tray as follows 1 Locate the two microplate rack positioning slots located on the spot cutter base plate just left of the gel tray see Figure 2 3 Note that one slot is shorter than the other Long Slot Short Slot Figure 2 3 Microplate rack positioning slots 2 Locate the microplate rack positioning tabs on the rack Note that the tabs on one side of the rack are more closely spaced than the tabs on the opposite side ya Widely spaced tabs Closely spaced tabs Figure 2 4 Microplate rack 3 Orient the rack for the type of microplates that will be used For standard microplates t
47. e water generated from washing gel tips The level sensor prevents experiments from being run until the waste is emptied Waste pump The waste pump is used to pump waste from the wash station waste basin see Figure 1 6 to the waste reservoir Waste filter The filter is used to prevent gel and membrane debris from clogging the waste pump This filter should be checked and cleaned periodically see Maintenance Section 10 1 Wash Station The wash station is used to reduce cross contamination between cuts After each cut the cutting head moves to a microplate well deposits the gel or membrane plug and then goes to either the gel tip wash station or the membrane tip wash station e Basin Gel Tip 5 Wash Port Jn eco MS gt Membrane Tip Wash Station Figure 1 6 Wash station INTRODUCTION 1 2 7 Gel Tip Wash Station The gel tip wash station is located on the left end of the wash station block lt consists of a gel tip wash port a waste basin and a waste level sensor The gel tip is cleaned as it is positioned in the wash port and water is pumped through the cutting tip and up around the tip s outer surface and then down to the waste basin The waste water level sensor detects when the basin is full and causes its contents to be pumped to the waste reservoir Membrane Tip Wash Station The membrane tip wash station consists of three wash ports in which three 1 5 ml capless tubes catalog 165 7212 are placed These tubes
48. ee Edi pte X Coce DI Heb Figure 8 9 Plates for Cut Run dialog box The Plates for Cut Run dialog is used to identify microplates to be used for a cut run Use The check boxes in this column are used to select or deselect plates for use in the current run Name This column is used to enter a user defined microplate name for each plate The software automatically names unnamed plates with the order they will be filled i e Plate001 Plate002 etc by default This name may be changed if desired Barcode Plate ID This field is used to enter the microplate barcode ID or a user defined microplate ID for each plate Although a bar code or plate ID is not required it is highly recommended since it can be used when loading plates to confirm that the correct plate has been loaded into the correct microplate rack position see Section 8 10 The barcode can be scanned in using a barcode reader or typed in from the keyboard Size This column lists the number of wells in the plate Order This column lists the fill order for each plate Avail This column displays the number of available wells in each microplate Used This column displays the number of wells that have been filled or reserved Edit Click these buttons to show an image of the selected plate see Figure 8 9 The microplate image displays the status for each well white empty well and red filled or reserved wells Delete Click the buttons in this column t
49. eft 4 clicks the front side of the ring will move to the left Adjust the focus upper ring on the camera lens while you monitor the image and the Focus Levels field in the camera focus dialog The Focus Levels will generally reach a minimum when the camera is focused When finished change the aperture to f stop 12 To set the aperture to f stop 12 turn the lower ring all the way to the right the front of the ring will move to the right and then turn it to the left 2 clicks the front side of the ring will move to the left The camera focus is now set to the optimum position Click Done EXQUEST CALIBRATION WIZARD ea eg ES j Cobo F bg Ss vee 09 59 Show gad on mapa Figure 3 5 Camera focusing dialog 6 Head Height Calibration The Head Height Calibration dialog is used to set the cutting tip height used for system position calibration and gel blot cutting a b C d Remove the calibration target from the cutting deck if it is present Place a gel cutting sheet in the gel tray and install the gel tray in the EXQuest Spot Cutter Check Lower head to adjust height to lower the cutting head to the gel tray Use the Raise Lower slider to adjust the tip height Raise the gel tip slightly above the cutting tray and then slowly lower it until it touches the cutting deck Continue lowering the tip until all the slack in the Z arm is taken up e g the tip can no longer be lifted off the cutting tray with mild force Th
50. ere should be no spring compression of the cutting head during this step Click Next il EXQuest Spot Cutter Setup dnjas S9NOXA EXQUEST CALIBRATION WIZARD Head Height Calibration Remove the focusing template if present Install the gel tray in the EXQuest Spot Cutter Position the head so it just touches the gel tray as shown There should be no spring compression however there should be enough downward force to take up the slack in the Z motion mechanism Lower I Lower head to adjust height Cancel Figure 3 6 Head Height Calibratioin screen 7 Flat Field Calibration The flat field calibration step corrects for inhomogeneity in the light source Before you start make sure the gel tray is installed and that it is clean and dry Click Next to proceed with the calibration Back to repeat the previous step or Cancel to abort the calibration T EXQuest Spot Cutter Setup Ei dnjag Jeenpvz Flat Field Calibration Remove the focusing template il present Disan the ight box surface and the gel tray Make sure the gel tray is properly installed in the ExQuest Spot Cutter as shown Close the door and cick Next lt Back Next gt Cancel Figure 3 7 Flat Field Calibration screen 3 5 EXQUEST CALIBRATION WIZARD Lens Calibration The lens calibration step corrects for image distortion due to the camera lens To perform the lens calibration place the Calibration Target on the gel
51. es As downward motion is applied to the cutting head the mechanism causes the tip to twist and cut the membrane For best cutting efficiency a sharp tip should be used If the tip becomes dull or damaged it should be replaced The membrane cutting tip must contact water to efficiently eject membrane plugs Therefore microplate wells and tubes must be filled with enough water to cover the end of the cutting tip Low water level in the wells will result in membrane plugs missing the well or being delivered to waste Caution the membrane cutting tips are sharp The membrane tips should only be used to cut PVDF and nitrocellulose membranes in order to maintain their sharpness New membrane cutting tips may be purchased from Bio Rad see Accessories Section 9 Fluidics System The fluidics system consists of water and waste reservoirs pumps and sensors It has two major functions gel spot pickup and delivery and waste removal 1 2 6 Syringe Pump The syringe pump delivers water used for gel plug delivery gel and membrane hydration and gel tip washing It is also used to create a weak vacuum for loading gel plugs into the cutting tip Water reservoir and level sensor This reservoir holds cross contamination water used for gel plug delivery gel and membrane hydration and gel tip washing The level sensor alerts the operator that the water level is low and stops the cut run Waste reservoir and level sensor This reservoir holds wast
52. est gel s to cut based on spot quantity and quality Spot quantity usually varies between gels in an analysis set In general you want to cut from gels where the spot quantity is higher to get more sample Step 2 Select Gets C Select from Est Auto select Tolerance 2 20 a Y 34 include quality Gels 2 Figure 6 24 Step 2 Select Gels Auto select b Set the Tolerance The tolerance value is used to determine the spot quantity range that is acceptable when choosing the gel from which a particular spot will be cut If the quantity of a spot in only one gel falls within the tolerance that gel will be auto selected for the spot cut However if the quantity of a spot in two or more gels falls within the tolerance range then the auto select mechanism will consider which gels will best minimize the time and effort required to switch gels on the spot cutter platform For example if you have five gels in an analysis set with the tolerance ratio set to 1 the lowest setting auto selection specifies different cuts in all five gels requiring you to change gels in the spot cutter five times However by increasing the tolerance ratio slightly auto select finds that three gels contain spots whose quantities fall within the specified tolerance and it adjusts the cuts so that only those three gels are selected This will save you considerable time and effort switching gels in the spot cutter As you adjust the tolerance monitor the number of
53. esume on the Manual Excision Tool screen see Step 17 above Optional After a cut run is complete you may use the Confirm cuts function to confirm that the cuts have been made This is generally not necessary due to the high cutting efficiency of the EXQuest spot cutter e Microplate Imaging If the gel image was acquired with the UV EPI light source a pop up box will be displayed at the end of the run that gives you the option to acquire a confirmation image of the microplates Click Yes to acquire a microplate image see Section 5 3 1 for more detail e Gel Imaging When a run is finished return to the Manual Excision Tool screen and click Confirm cuts to take a gel confirmation image This image can be used to determine if all the selected spots have been cut from the gel see Section 5 3 2 for more detail Press Save to save the gel image as well as the cut request list Microsoft Excel formatted table This cut list can be imported back into the Manual Excision Tool for use when cutting additional gels in the same set see Section 5 2 Click Close to exit the Manual Excision Tool screen IMPORTING A CUT LIST The Import function is used to import spot coordinates from a tab delimited text file These files can be generated from previous Manual Excision Tool cut runs or from non Bio Rad image analysis software You may only import a cut request list if the current image does not currently have cut requests associated with i
54. f gel to cut see Section 8 4 e Free Gels Select this option to use a protocol optimized for cutting non backed gels e Plastic backed Gels Select this option to use a protocol optimized for cutting plastic backed gels When this option is used gel plugs are cut and then dislodged from the gel backing by a side to side cutting tip motion e Glass backed gels Select this option to use a protocol optimized for cutting glass backed gels When this option is used gel plugs are cut and then dislodged from the gel backing by a side to side cutting tip motion Note that the cutting tip is raised by 3 mm to accommodate the glass plate e PVDF This option is available only when the spot cutter is in nitrocellulose membrane cutting mode and is used to specify that either a PVDF or Nitrocellulose membrane will be cut When cutting blots Fill microplate wells with DI water to within 1 to 2 mm of the top In order to release the cut membrane into a microplate well the cutting tip must be in contact with water Do not overfill the wells tubes as this will allow cut membrane to float between adjacent wells Optional Select Make multiple cuts on large objects in the Cut Options panel see Section 8 4 to harvest large amounts of protein from a single spot and deposit it into a single microplate well Enter the maximum number of cuts that you want to allow in the Maximum cuts object field Select the microplate format to be used from t
55. for the number of cuts selected the number of plates required and the required plate well volume MANUAL EXCISION TOOL 17 18 19 20 21 22 5 2 Click Begin Resume to open the Plates for Cut Run dialog see Section 8 9 and then enter the plate name and barcode plate ID in their respective fields The plate barcode can be entered using a barcode scanner or typed in To reserve wells on a microplate click the Edit button for the desired microplate and click on individual wells to reserve them Press Done to open the Load Plate on Cutter dialog box see Section 8 10 Locate the microplate with the name and barcode plate ID shown in the dialog and then scan or type in the barcode plate ID into the Confirm barcode field If the Confirm barcode does not match the Barcode a warning will pop up that states there is a barcode mismatch In this case make sure that you have the correct microplate and have entered the barcode correctly Place the plate in the microplate rack position identified by the green box with well A1 located in the front left plate corner Click Done to proceed to the next plate or Prev to return to the previous plate Click Finish to start the run If at any time the cut run needs to be paused or aborted press Pause This will cause the cut run to pause after the current cut To abort the run click Cancel to resume the run click Resume After aborting a run it can be restarted by pressing Begin R
56. fter each cut the cutting tip is cleaned as it is dipped into the tubes of DI water If desired review the cut request list see Figure 6 9 8 ANALYSIS SET EXCISION TOOL C Analysis Set Excision Control Tool Cus bt Analysis vet mame Sample Anatysie Set 4 Gel spots to cut 147 Anaita vet ipot court 49 Total cuts fei m i 147 Cu MIP SSP Gel Cut dato Stan a 1410 en tanpe 3 1406 1502 tie fg 1402 0 somete i 1 AD tange i 3 Am 702 tangle Ed 1403 angie D 1 405 sange IS 1404 tamgie 3 1500 rampie fg on tan ie Custer opens Wath optens Mpchote Wath volume 50 0 u Ed Cow G Hep Figure 6 9 Analysis Set Excision Control Tool Analysis set name This text field displays the name of the selected analysis set Analysis set spot count This text field displays the number of spots contained in the analysis set Gel spots to cut This text field displays the number of analysis set spots selected to be cut Total cuts incl multi This text field displays the total number of cuts that will be made This value is always greater than or equal to the Gel spots to cut value and depends on the Maximum cuts Object value as well as whether protein spots are being pooled Cut List Table This table provides information about each spot that will be cut Rows represent individual spots and columns display information about each spot Cut Check this box to cut or re cut the spot MTP Displays the microplate number
57. ftware version information WARRANTY 12 0 WARRANTY EXQUEST SPOT CUTTER WARRANTY INFORMATION This warranty statement may vary outside of the continental United States Please contact your local Bio Rad office for the exact terms of your warranty Bio Rad Laboratories warrants to the customer that the EXQuest Spot Cutter System Catalog number 165 7200 or 165 7201 will be free from defects in material and workmanship and will meet all of the performance specifications for a period of one year from the date of shipment This warranty covers all parts and components of the spot cutter except those such as calibration targets and pucks gel and membrane cutting sheets cutting tips and other items where normal wear requires frequent replacement This warranty does not cover items broken or damaged by improper handling or use If any defects should occur during this period Bio Rad Laboratories will either replace or repair the defective parts free of charge For the exact terms of warranty please see the Instrument Warranty Card In the event that the system must be returned to the factory for repair under warranty the instrument must be packed and returned in its original shipping container If this is not available contact Bio Rad Technical Support for instructions Bio Rad shall not be liable for any incidental special or consequential loss damage or expense directly or indirectly arising from use of the EXQuest Spot Cutter system Bio Rad
58. ge spots depends on both the Maximum cuts object value and the size of the box drawn around the spot Cutting tip size Use this control to configure the software for use with the cutting tip 1 0 mm or 1 5 mm installed on the spot cutter EXCISION TOOL CONTROLS Material Use this drop down menu to select the gel type that will be cut e g Free gel Plastic backed gel or Glass backed gel When the membrane head is installed PVDF will automatically be selected 8 5 PLATE OPTIONS PANEL Specify Options Cut Plate Hydration Wash 96 wellMTP 0 15 _y ml per well 384 well MTP 0 05 96 Tubes 1 00 ml per well G G ml per tube Loading order 41 42 C 41 B1 Figure 8 5 Plate Options panel The Plate Options panel is used to set the microplate format well volume and the well loading order This panel may appear in a tab box as shown above or as a separate panel 96 well microtiter 384 well microtiter 96 tubes Use these option buttons to select the plate format to use ml well and m tube These drop down menus are used to identify the well volume for the microplate format selected Note that ml well identifies the actual microplate well volume whereas Min Well Volume in the Run Info panel Section 8 8 is the minimum required well volume and is based on the Maximum Cuts object value and any pooling Analysis Set Excision Tool advanced mode only performed
59. gels selected by the tolerance setting c Check the Include quality box to consider spot quality when auto selecting spots When this checkbox is selected a well resolved spot of sufficient quantity will be favored over a less well resolved spot of higher quantity within the specified tolerance ratio After checking the Include quality box look to see if it has changed the number of gels selected from which cuts will be made 6 15 ANALYSIS SET EXCISION TOOL 12 Optional Click the Review tab and review the cut selections The Review tab displays the gel spots by Standard Spot Number SSP along with their intensity in each gel In the table columns represent gels and rows represent spots The cut requests are highlighted in blue The table will initially show one cut request per spot that is one highlighted blue cell per row JEnable editing by clicking on cells below to select deselect spots to cut 291 sample3 samplel sample2 0001 196519 4 0101 160361 0 195664 1 0201 121381 2 0301 437086 2 340417 5 0501 0502 0503 0504 0601 Totals 476117 3 3092529 264966 9 241357 1 208765 1 201385 9 108218 3 242280 7 94188 2 178384 3 24 180 lo EE D Figure 6 25 Review table If after reviewing the cut selections you wish to add or remove cuts click on the Enable editing by clicking on cells below to select deselect spots to cut check box and then click on the indi
60. he Plate Options panel see Section 8 5 and then specify the well volume and well loading order The well volume will determine the maximum number of spots that can be placed in a single well tube MANUAL EXCISION TOOL 12 Optional Check Hydrate in the Hydration Options panel to automatically hydrate the gel during the cut run Specify how often it is to be hydrated see Section 8 6 then click Define hydration area to identify the gel or blot location Gels and membranes should typically be hydrated at least once every hour 13 If gels are being cut enter the wash volume required to clean the gel cutting tip 50 to 500 ul see Section 8 7 in the Wash Options panel 14 Add a cut request to the cut list Cut request can be added manually as shown below or by importing them from a tab delimited text file see Section 5 2 a Check Snap to peak in the Specify Cuts panel to activate a pixel peak finding function that centers the cut request on a spot s darkest pixel or leave it unchecked to center the cut circle at the location where the mouse was clicked Specify Cuts Ye Addimowe cut Lo Remove cut Snap to peak Figure 5 3 Specify Cuts panel b Zoom into the area of interest c Click Add move cut to activate the Add move cut tool d Click on each spot to be cut If you checked Make multiple cuts on large objects in Step 10 above you may click and drag a box around large spots to make a multiple cut request Th
61. he microplate rack is oriented shallow side up as shown in Figure 2 4 above For deep well plates and 96 tube racks the microplate rack is oriented deep side up 4 Place the rack into the spot cutter so that the closely spaced tabs are inserted into the short slot and the widely space tabs are inserted into the long slot Slide the microplate rack in the slots until you feel it drop into place 2 4 SYSTEM INSTALLATION Widely _ Closely spaced tabs spaced tabs d d Figure 2 5 Installed microplate rack 2 6 CABLE SETUP Before the spot cutter can be started the power cord and USB cable must be connected 1 Connect the square end of the USB cable to the EXQuest spot cutter hub labeled Host PC located on the instrument right side see Figure 2 6 below 2 Connect the other end of the USB cable to any USB port on the host computer 3 Connect the AC power cord to the EXQuest spot cutter power entry module located on the instrument right side 4 If an accessory bar code reader will be used with the spot cutter plug it into one of the peripheral USB ports on the spot cutter or computer 2 5 SYSTEM INSTALLATION USB USB to Fuse AC ports Host PC power Figure 2 6 Spot cutter USB ports and power entry module 2 7 CUTTING HEAD INSTALLATION There are two types of cutting heads available for the EXQuest spot cutter gel head standard and membrane head optional The gel head is supplied with both 1 0
62. he cutter image spots do not align correctly make sure that the file size is set correctly on the device used to scan the analysis set image For example the FX Densitometer and the GS series automatically set file sizes whereas camera scanners cannot If your analysis set image was generated from a camera acquisition device such as the Gel Doc EQ ChemiDoc EQ or VersaDoc it will not have specified file dimensions For such images you will need to enter the physical size dimensions correctly at the time of acquisition It is important to have correct physical dimensions before you proceed with the spot detection and match set creation otherwise auto alignment will not work correctly Manual Alignment Manual alignment positions cut marks at the location defined by the green data area box and in the same relative positions that the spots appear in the analysis set image It does not adjust the cut positions to optimize the fit Manual alignment should be done when there are only a few spots to align Set the Confidence threshold to an acceptable level We recommend it to be a high value Cut marks will be colored green for spots that have an alignment confidence greater than the Confidence threshold or colored red for those spots that have an alignment confidence below the confidence threshold Step 3 Confirm alignment 2 Jump and confirm Confidence threshold te Cuts below threshold 3 Total cuts 114 Figure 6 15
63. heet on it and then transfer the gel onto the cutting sheet Alternatively use the cutting sheet to lift the gel out of its storage container and then place both the cutting sheet and gel on the gel tray A cutting sheet should always be used to cover and protect the gel tray from being scored by the cutting tip Do not remove the thin plastic film from the cutting sheet as it is required for high cutting efficiency 3 Position the gel s in the center of the tray 4 Clamp the gels and cutting mat in place with the gel clamps see Figure 4 1 Use two clamps to hold a single gel in place or 2 to 4 clamps to hold 2 to 4 gels or broken gels in place The gel clamps are installed by inserting the them into the cutting tray slots at the front and back of the gel tray See Figure 4 1 The spring clip should be oriented to the front The gel clamps should be slid tightly up against the sides of glassed backed gels or directly on all other types of gels and blots Figure 4 1 Gel placement for A Single gels B Multiple gels al Insert the gel tray into the spot cutter and make sure it is latched securely see Section 2 4 by pushing toward rear until it snaps in place 4 2 PLASTIC AND GLASS BACKED GELS 1 Store gels in a container that keeps them protected and hydrated until needed for spot cutting Use the gel manufacturer s recommended storage conditions Dry the cutting sheet surface as well as the back side of the plastic or glass ba
64. ick Begin Resume Cuts to perform the re cuts 5 3 2 MANUAL EXCISION TOOL Identifying Re cuts from a Gel Image When the cut run is finished return to the Manual Excision Tool screen and click Confirm Cuts to acquire a confirmation image Closely examine the gel image for spots that were not picked up Use the zoom and transform functions on the toolbar to get a better view of the gel When a missed spot is located click in the Manual Excision Tool window and then right click on the missed cuts cut circle to the recut it Switch back to the Confirm Cuts image and repeat steps 2 and 3 until all missed spots are selected to be re cut Click Begin Resume Cuts to perform the re cuts ANALYSIS SET EXCISION TOOL 6 0 ANALYSIS SET EXCISION TOOL The Analysis Set Excision Tool is designed to use the advanced capabilities of the PDQuest software in creating cut request lists and cutting gels and blots With the Analysis Set Excision Tool you can manually select the gel or gels contained in an analysis set to cut or let the software automatically select the best gels and spots in an analysis set to cut 6 1 SETTING UP AN ANALYSIS SET EXCISION RUN 1 Start PDQuest software and open the match set that contains the desired analysis set To create an analysis set see the PDQuest user manual 2 Click Identify Analysis Set Excision Tool see Figure 6 1 to open the Analysis Set Excision Wizard Report Window Help Analysis
65. in 0 2 mm increments or click and drag the slider to make larger adjustments c When the tip is centered check Lower head to adjust eject height to lower the cutting tip into the well Use the sliders to center the tip in the well T ExQuest Spot Cutter Setup y x Plate Position Calibration Postion the head over woll Al of the right reat Container as shown It should be centered over the well and the tip when lowered should be slightly above the well Lett A A Front vk Bech dnjas sanox3 lt Back Next gt Cancel Figure 3 11 Plate position calibration cutting tip adjustment 3 8 EXQUEST CALIBRATION WIZARD Use the Raise Lower slider to adjust the tip height Typically the gel tip should be placed about O to 1 mm into the well and the Membrane cutting tip 1 2 mm into the well Note that the membrane cutting tip must be in contact with water in the wells to release membrane plugs To ensure that the cutting tip touches water when releasing the membrane plugs fill the microplate with the amount of water that will be used during the cut run and then calibrate the microplate tip height so it just touches the water Press Next to save the calibration To calibrate another plate format click Yes and then Next see Figure 3 12 To continue to the Wash Station Position Calibration step click No and then Next Press Cancel to quit the calibration The calibration completed in all previous steps will be saved
66. l head Last calbeated on 03 Jan 2005 16 00 You wil need the calibeation gid template and 9 calibration pucks It you have not aignad and locused the camera place the focusing template on the gel tray insert the tray into the EXDuest SpotCutter and check the box below _ Perform focus abgnment step C Skip to plate position step Click Newt when ready dnjas sanox3 Figure 3 4 EXQuest Calibration Wizard screen calibrated From this screen you may Calibrate the camera with the current focus and aperture setting by clicking Next Go to Step 6 Head Height Calibration Focus the camera and then calibrate it by checking Perform focus alignment step and then clicking Next go to Step 5 Camera Focus Skip the camera calibration steps by checking Skip to plate position step and then clicking Next go to Step 9 Plate Position Calibration 5 Camera Focus The Camera Focus dialog is used to monitor a camera image as the camera is focused a b With the gel tray inserted in the spot cutter place the calibration target on the gel tray and align it with the left and front sides of the tray Locate the camera lens inside the spot cutter above the door in between the UV lamps and change the aperture lower ring on the lens to f stop 6 To set the aperture to f stop 6 turn the lower ring all the way to the right the front of the ring will move to the right and then turn it to the l
67. land 09 804 22 00 France 01 47 95 69 65 Germany 089 318 84 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 455 8800 India 91 124 2398112 3 4 5018111 6450092 93 Israel 03 951 4127 Italy 39 02 216091 Japan 03 5811 6270 Korea 82 2 3473 4460 Latin America 305 894 5950 Mexico 55 52 00 05 20 The Netherlands 0318 540666 New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 095 721 1404 Singapore 65 64153188 South Africa 00 27 11 4428508 Spain 34 91 590 52 00 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 2578 7241 United Kingdom 020 8328 2000 Sig 1204 10001531 RevA
68. le design The gel tray can be easily removed from the spot cutter so that gels and blots can be placed on it from the convenience of the lab bench Gel Clamps The gel tray includes clamps used to hold gels and blots securely in place Protective Sheets The disposable gel cutting sheets patent pending are used to protect the gel tray surface from damage by the cutting tips see Accessories Section 9 for ordering information Failure to use the protective cutting sheets will damage the gel tray and void its warranty Cutting sheets can also be used to lift gels out of storage trays to minimize damage from excessive handling 1 3 INTRODUCTION Figure 1 4 Gel tray with clamps 1 2 4 Cutting Heads The EXQuest spot cutter supports both gel and membrane blot cutting heads The gel head is supplied with 1 0 mm and 1 5 mm diameter cutting tips The membrane head is supplied with 1 0 mm diameter cutting tips Figure 1 5 EXQuest membrane head left Patent pending and gel cutting head right e Gel Cutting Head The gel cutting head is furnished as standard equipment and consists of a stainless steel gel tip 1 0 mm or 1 5 mm that is spring loaded into a Delrin bracket see Figure 1 5 The assembly should be replaced if it is damaged The needle does not require sharpening 1 2 5 INTRODUCTION Membrane Cutting Head The membrane cutting head is an optional accessory that is used for cutting PVDF and nitrocellulose membran
69. ls and then click Next to cut previously cut spots from uncut gels in the analysis set go to Create a cut request list above Select Re cut existing finished cuts and then click Next to re cut previously cut spots Go to Section 6 2 Setting up a Cut Run Select the gels to cut Manual Gel Selection ANALYSIS SET EXCISION TOOL T Analysis Set Excision Wizard Select Gels to Cut You may select one or more gels from which to cut spots if you select more than one gel then cuts taken from matched spots will be placed into the same well to obtain more of the protein Gel Spotd Cuts E samples vt 204 0 E sample v2 se e sample vi 112 0 e UOISIDX3 Joe sisAjeuy Figure 6 6 Select Gels to Cut Screen From the Select Gels to Cut screen select one or more gels to cut Multiple gels may be selected by Ctrl click or Shift click Click Next when finished In the Select Gels to Cut screen the Spots column lists the number of spots for each gel in the analysis set and the Cuts column shows the number of cuts that have been selected from each gel In Manual Gel Selection all the spots in a selected gel will be cut If more than one gel is cut all the spots with the same Standard Spot SSP number will pooled together to increase protein yields e Automatic Gel Selection Analysis Set Excision Wizard Automatically Select Gels to Cut This wizard will help you determine the fewest number of gel
70. luor S MAX x ChemDoc EQ 5 Fluor S MAX2 x ChemiDoc XRS VersaDoc 2 65 700 5 Personal PX 165 710 FX X 65 800 Display Toolbaes Appbcation Devices Security Spot cutter C Not set EXQuest Spot Cutter ProteomeWorks Pius Spot Cutter 30cm Proteome orks Spot Cutter 25cm Analysis Set Excision Tool advanced user mode Y 0 X Cancel DI Help Figure 6 20 Preferences Dialog Box 6 13 ANALYSIS SET EXCISION TOOL 10 11 6 14 Check Analysis Set Excision Tool advanced user mode in the Spot cutter panel and then click OK Make sure the spot cutter is set up as described in Sections 2 and 3 and that the required cutting head is connected to the spot cutter Prepare the gel or blot as described in Section 4 Make sure that the DI water bottle is full and the waste bottle is empty at the start of the run If cutting blots fill three 1 5 ml capless microtubes with water to approximately 1 mm below the tube top and then place the tubes into the three membrane tip wash ports After each cut the cutting tip is cleaned as it is dipped into the tubes of DI water Start PDQuest and open the match set that contains the desired analysis set To create an analysis set see the PDQuest software user manual Click Identify Analysis Set Excision Tool see Figure 6 21 to open the Analysis Set Excision Wizard yze Report Window Help Analysis Set Excision Tool Manual Excision Tool
71. mm and 1 5 mm inner diameter nominal cutting tips The membrane head is supplied with a 1 0 mm cutting tip 2 7 1 Gel Cutting Head Figure 2 7 Gel cutting head installation SYSTEM INSTALLATION To install or change the gel cutting head Open the front door by lifting it all the way up and then lowering it until it latches 2 Press the Change Cutting Tip button located on the lower right hand side of the bezel This will cause the cutting head to move to the front of the cutting tray for installation 3 Ifthe membrane cutting head is installed unplug its electrical connector from the PC board located on the robot arm 4 Unscrew the knurled thumbscrew on the robot vertical arm to disconnect the currently installed gel cutting head or membrane head 5 Disconnect the 14 28 fitting by hand or use the V4 28 fittings wrench if needed 6 By hand or with the aid of the fittings wrench connect the 1 8 in tubing to the cutting head by way of its 4 28 fitting Use the supplied 1 4 5 16 open and wrench to prevent the gel tip from rotating 7 Attach the gel cutting head to the robot with the knurled thumb screw included with the spot cutter 8 Make sure the 4 28 fitting is snug but do not over tighten 9 Tighten the knurled thumbscrew 10 To close the door lift it slightly and then pull it down until it is closed 2 7 2 Membrane Cutting Head Ws Figure 2 8 Membrane cutting head installation To install a membra
72. n 8 4 Note that the cut request circle size displayed on the image depends on the tip size selected 14 Specify the type of gel to cut see Section 8 4 in the Cut Options panel e Free Gels Select this option to use a protocol optimized for cutting non backed gels 6 16 15 16 17 18 19 ANALYSIS SET EXCISION TOOL e Plastic backed Gels Select this option to use a protocol optimized for cutting plastic backed gels When this option is used gel plugs are cut and then dislodged from the gel backing by a side to side cutting tip motion e Glass backed gels Select this option to use a protocol optimized for cutting glass backed gels When this option is used gel plugs are cut and then dislodged from the gel backing by a side to side cutting tip motion Note that the cutting tip is raised by 3 mm to accommodate the glass plate PVDF This option is available only when the spot cutter is in membrane cutting mode and is used to specify that either a PVDF or nitrocellulose membrane will be cut Tips for cutting membranes Fill microplate wells with DI water to within 1 to 2 mm of the top In order to release the cut membrane into a microplate well the cutting tip must be in contact with water Do not overfill the wells or tubes as this will allow cut membrane to float between adjacent wells Optional Select Make multiple cuts on large objects in the Cut Options panel see Section 8 4 to harvest large amo
73. ne cutting head 1 Attach the 1 0 membrane cutting tip to the membrane cutting head Two wrenches spanners have been included with the membrane cutting head for this purpose First install the cutting tip by screwing it all the way on and then tightening the lock nut to hold the tip in place Caution the membrane cutting tips are sharp 2 Connect the membrane cutting head to the robotic arm with the 8 32 capscrew included with the membrane head Tighten the capscrew with a 9 64 hex wrench 3 Plug the membrane cutting head power lead into its electrical connector The connector is located on the circuit board adjacent to the membrane cutting head solenoid Plug the power lead into its connector as shown below in the proper orientation i wll tab and groove _ gt H Insert Connector tik Figure 2 9 Membrane cutting head electrical connector 4 Connect the 1 8 inch tubing by way of its 1 4 28 connector to the female connector located on the metal block above the cutting head attachment point 2 7 SYSTEM INSTALLATION 5 Insert three clean 1 5 ml capless tubes into the membrane tip wash port slots located on the wash station s right side Fill these tubes with water to approximately 1 mm below the top 2 8 DI WATER AND WASTE BOTTLE INSTALLATION Figure 2 10 DI water and waste bottle installation Two 1 liter bottles have been supplied with the EXQuest spot cutter for use as DI water and waste
74. ne of the three tubes to wash the tip and remove any membrane plugs that have adhered to the tip 6 Inthe Acquire Image panel see Section 8 1 select White Trans for visible stained gels such as Coomassie or silver stain or UV EPI for fluorescent stains such as SYPRO Ruby MANUAL EXCISION TOOL 10 11 5 2 z Manvel Lucicion Tool connected to XQuest with Gel Head simulated Icom mayo Specs Opiicra oe Pate Pomon Wah Mabe mape OF when pur fe Memm atid 9 D r ne 10m Clie Meng free a e Specs D Y Addimows cu Rot teg Ee kto D r 20 Ma wel M ons Web 727 Pise 3 Stow Tee Peston pres DH 7a Ql ws Dy DATE T pe puro a DD Cue HI He T Teron Figure 5 2 Manual excision tool Close the spot cutter front door and click Acquire Image to take an image of the gel tray contents Do not open the door during image acquisition As a safety precaution the UV EPI lights will automatically turn off if the door is opened Images acquired with UV EPI are automatically inverted by the software to display dark spots with a light background If the image does not detect enough spots or is too faint use the transform function to optimize the image contrast see Section 8 3 Identify the cutting tip size 1 0 mm or 1 5 mm in the Cut Options panel see Section 8 4 Note that the cut request circle size displayed on the image depends on the tip size selected From the Cut Options panel specify the type o
75. nned gels can be cropped rotated etc with the image editing controls With Automated Spot Detection and Matching you can select the gels you want to analyze detect spots of interest and create match sets and an analysis sets The Analysis Set Excision Tool is used to determine which gel s in an analysis set are best to cut from and then to cut the gels The Analysis Set Excision Tool is used to cut one large gel or up to four small gels blots without the need to swap gels during the cut run See Section 6 Analysis Set Excision Tool for more details Quantity One Software The full featured Quantity One software provides additional capabilities that include image analysis and lane volume band excision In addition Quantity One can acquire gel images using any of several Bio Rad imaging systems These scanned gels blots can be cropped rotated etc using the image editing controls See Section 7 Lane Volume Excision Tool for more details 1 2 12 Optional Barcode Reader The barcode reader when purchased can be used to record microplate or gel barcodes with the cut run data Its use is optional but is recommended when working with large numbers of microplates to avoid mixing plates up See Sections 8 9 and 8 10 for use with the PDQuest or Quantity One software The barcode reader can be plugged into any one of the USB ports on the spot cutter see Section 2 6 or computer For more information see the instructions shipped with the barcode
76. o delete a plate from the list Add plate Click this button to add a new plate to the list EXCISION TOOL CONTROLS 8 10 LOAD PLATE ON CUTTER DIALOG 4 Load Plate on Cutter Load PlateD01 into the front night position Name Page Figure 8 10 Load Plate on Cutter dialog box This dialog helps the operator correctly place the microplates in the microplate rack e Name This is the name of the microplate to be loaded Barcode This is the barcode or plate ID of the microplate to be loaded e Confirm barcode This is the barcode or plate ID of the microplate you are loading and is scanned or typed in to confirm that the correct plate is being loaded Failure to use a barcode plate ID could result in a plate being placed in the wrong rack position A barcode or plate ID both instances must be entered if one was entered in the Plates for Cut Run dialog see Section 8 9 e Plate type This field displays the microplate format for the microplate currently being loaded e Microplate Rack Image The green box in this image shows the rack location that the current microplate should be placed in Note that the microplate should be oriented so well A1 is in the front left corner ACCESSORIES 9 0 ACCESSORIES SUPPLIES REPLACEMENT PARTS Catalog Number Description 165 7208 EXQuest Cutting Sheets S pkg of 15 165 7205 EXQuest Calibration Pucks R pkg of 10 165 7209 EXQuest Gel Clamps R pkg of 4 165 7202 EXQuest
77. oomed image to full view Drag amp Zoom Click this button to zoom into an area defined by click and drag of the mouse ls a e Drag Tool Click this button to activate the drag hand cursor and move the zoomed image to an adjacent area LS Zoom In Click the Zoom In button place the cursor over the area to zoom in on and then click the mouse The image will be zoomed in centered at the cursor location Click in the zoomed image to zoom in further p Zoom Out Click this button to activate the Zoom Out cursor and then place the cursor over the image and click the mouse Click in the image again to zoom out further E EZ Transform This image control button is used to open the Transform dialog see Section 8 3 EXCISION TOOL CONTROLS 8 3 TRANSFORM DIALOG BOX The Transform dialog box is used to adjust the image brightness and contrast lt contains controls used to view the pixel intensity frequency distribution manually or automatically transform the image check for saturated pixels or color the image il Transform sample1 v1 Raw 2 D Image Note that transform operations change only the view of the data not the data itself Transform Plot Transform Plot Preview Window hubo A Frequency Distribution Full scale Low High jLog 4 Frequency Distribution Histogram Display Invert display Log High Low Sliders i Highlight saturated pixels Always auto scal
78. option to use a protocol optimized for cutting unbacked gels Plastic backed Gels Select this option to use a protocol optimized for cutting plastic backed gels When this option is used gel plugs are cut and then dislodged from the gel backing by a side to side cutting tip motion Glass backed gels Select this option to use a protocol optimized for cutting glass backed gels When this option is used gel plugs are cut and then dislodged from the gel backing by a side to side cutting tip motion Note that the cutting tip is raised by 3 mm to accommodate the glass plate ANALYSIS SET EXCISION TOOL 10 11 12 13 e PVDF This option is available only when the spot cutter is in membrane cutting mode and is used to specify that either a PVDF or Nitrocellulose membrane will be cut When cutting blots Fill microplate wells with DI water to within 1 to 2 mm of the top In order to release the cut membrane into a microplate well the cutting tip must be in contact with water Do not overfill the wells tubes as this will allow cut membrane to float between adjacent wells Optional Select Make multiple cuts on large objects in the Cut Options panel see Section 8 4 to harvest large amounts of protein from a single spot and deposit it into a single microplate well Enter the maximum number of cuts that you want to allow in the Maximum cuts object field Select the microplate format to be used from the Plate Options panel
79. p 2 Examine Image Make sueo that you can cee a visible gel plug m each well used Step J Select Action Dick on Specty ne cuts below to repeat any cuts The mage of the microtiter plate wil remain on the s cen dang meat selection ao Spectysecuts A Norecuts Figure 5 7 Image Microtiter Plate window Without moving the gel place a clean plastic cutting sheet on top of the gel Place the microplates on top of the plastic sheet note the orientation so you can recognize the A1 well location and then close the door Click Acquire image to obtain an image with the same exposure time as the cut request image Closely examine the microplate wells in the image for the absence of gel plugs and note the well position Use the zoom and transform functions See Section 8 3 on the right side of the Image Microtiter Plate window to get a better view of the microplate wells Click Specify re cuts to re cut spots or No re cuts if there are not any missed spots Click OK on the Remove Microtiter Plate pop up box and then return each microplate to its original location and orientation in the microplate rack Carefully remove the cutting sheet from the top of the gel so as to not move the gel If Specify re cuts was selected in Step 7 click OK and then find the missed spot pickups on the gel image from their well ID and right click on the spot to add it to the re cut list Press OK to close the Re cut request dialog Cl
80. r aborted press Pause This will cause the cut run to pause after the current cut To abort the run click Cancel to resume the run click Resume After aborting a run it can be restarted by pressing Begin Resume on the Lane Volume Excision Tool window EXCISION TOOL CONTROLS 8 0 EXCISION TOOL CONTROLS This section describes the excision tool controls that are common between the Manual Excision Tool Analysis Set Excision Tool and Lane Volume Excision Tool All other controls and dialogs are discussed in their respective sections 8 1 ACQUIRE IMAGE PANEL Acquire Image White Trans UY EPI CR Figure 8 1 Acquire Image panel fh Acquire image The Acquire Image panel is used to acquire gels and blot images 1 Select a light source for image acquisition e White Trans Select this option for visibly stained gels or blots e UV EPI Select this option for gels and blots stained with fluorescent dyes such as SYPRO Ruby The maximum exposure time is 10 seconds 2 Click Acquire image to obtain an image with the selected light option 8 2 IMAGE CONTROLS PESE Figure 8 2 Image controls Image controls are used to zoom in zoom out drag or transform gel and blot images They are located below or to the side of an image as well as on the PDQuest and Quantity One software s toolbar The image controls perform the following functions Full View This image control button is used to return a z
81. rating each half opening it up removing the metal screen and rinsing the assembly under water and then reassembling it e The outside of the spot cutter may be cleaned with water or mild detergent A paper towel wetted with ethanol may be used to wipe stains off the outside of the spot cutter however the excess ethanol should be wiped off immediately with a dry paper towel e The transilluminator should be cleaned routinely with a paper towel wetted with water or a mild detergent to remove all particulates that may interfere with the camera image 10 2 UV LIGHT REPLACEMENT The UV EPI lamp should typically be replaced once a year depending on its level of use To monitor the lamp life open the UV lamp log file SC_UVLightLog txt in the directory Documents and Settings All Users Application Data The Discovery Series install prm If the UV light intensity has dropped in half from the initial log entry the UV EPI lamps should be replaced as described below After installing the new lamps delete or move the file SC_UVLightLog txt so a new log file can be created The two fluorescent UV light fixtures are located above the cutting deck and each contains two fluorescent bulbs See Accessories Section 9 for ordering information All four fluorescent tubes must be replaced at the same time To replace 1 Use a Phillips screwdriver to remove the three screws from the front of the EPI lamp housing Figure 10 1 UV EPI lamp 2 Pull the UV EP
82. ratio slightly auto select finds that three gels contain spots whose quantities fall within the specified tolerance and it adjusts the cuts so that only those three gels are selected This will save you considerable time and effort switching gels in the spot cutter As you adjust the tolerance monitor the number of gels selected by the tolerance setting Check the Include quality box to consider spot quality when auto selecting spots When this checkbox is selected a well resolved spot of sufficient quantity will be favored over a less well resolved spot of higher quantity within the specified tolerance ratio After checking the Include quality box look to see if it has changed the number of gels selected from which cuts will be made 6 Click Next to Specify the Excision Options Analysis Set Excision Wizard Specify Excision Options X Make multiple cuts when possible Bwl MIP 0 15 mi per well Manamum cuts obyect E C Siwe MTP 005 miper wel Cutting tp size 1 0mm 1 5mm CI Tubes 100 y mipertube Material Free gel Loading order Gi A3 LI C A1 B1 Cuts 233 Min well volume 0 15 mi Wels 87 page Ent time gt S A Pa un Go so 2 A o 3 Figure 6 8 Specify Excision Options Screen 7 Identify the cutting tip size 1 0 mm or 1 5 mm in the Cut Options panel see Section 8 4 8 Specify the type of Material to cut see Section 8 4 in the Cut Options panel Free Gels Select this
83. reader 1 9 SYSTEM INSTALLATION 2 0 SYSTEM INSTALLATION Before the EXQuest spot cutter can be used the software must be installed and the instrument set up and calibrated At the time of installation a Bio Rad service representative will setup the instrument install PDQuest and or Quantity One software and then calibrate the instrument for you 2 1 PRE INSTALLATION REQUIREMENTS Before installing the EXQuest spot cutter ensure that the following requirements have been met Installation Requirements Checklist Bench Space e The EXQuest spot cutter should be placed on a sturdy lab bench Tables are not recommended but may be used if they are made of heavy materials and are anchored to the floor The EXQuest spot cutter requires a bench top area that is 36 inches 91 cm wide 27 inches 69 cm deep and 26 inches 66 cm high There should be 38 inches 96 cm of clearance above the front of the bench to allow the spot cutter door to open upward There must be at least 10 inches of free space on the left side of the spot cutter for access to the water and waste bottles There must be at least 12 inches of free space on both the left and right side of the spot cutter during installation e Space for a computer with monitor Power e A dedicated 20 Amp electrical circuit and two receptacles Nominal input voltage range 100 V to 240 V 50 Hz 60 Hz Power consumption 220 W e Surge protector Or Uninterruptible Power Supply
84. reen Jump ard conten 9 Delay cropped mage CIA marks JOA sn E pe A Ato Matches Multi vie Cetchere reteni Comente out overtags i 4 er ai ES cia 8 B J Let HALO Souen Data box Y aj gt Q Marne sl Cuts below threshold 0 Total cute 87 O roceed X Carcel O Heb Figure 6 12 Align Analysis and Cutter Images dialog box Spot Alignment Procedure a Click and drag the green data area box to position it over the gel image The green data area box is displayed when the Align Analysis and Cutter Images window is first opened or after Reposition is clicked in the Step 1 Place data box panel If the gel orientation is incorrect you may use the flip and rotate buttons to reorient the cutter image Step 1 Place data box E Reposition Flip Horizontal ji TW Rotate 180 Flip Vertical Rotate 90 Counter Clockwise Figure 6 13 Align Analysis and Cutter Images Step 1 Place Data Box Panel b Align the cutter image spots with those in the analysis set image Click Auto to have PDQuest software automatically align the image spots or Manual to manually align the spots ANALYSIS SET EXCISION TOOL 6 10 Step 2 Perform alignment El Auto El Manual Figure 6 14 Align Analysis and Cutter Images Step 2 Perform Alignment Panel e Automatic Alignment Automatic alignment uses a Gaussian fitting algorithm to align the cutter image spots with those in the analysis set image If t
85. reservoirs 1 Install the bottle holder Open the door on the left side of the spot cutter Unscrew the two thumb screws on the door and then mount the bottle holder on the door so that the two raised mounting pins are located in the slots on the top of the holder 2 Fill one bottle with DI water label it and then insert it into the front bottle holder slot 3 Label the second bottle Waste and then insert it into the rear bottle holder slot 4 Insert the water level sensor long sensor into the DI water bottle and the waste level sensor short sensor into the waste bottle 2 9 SOFTWARE INSTALLATION The following procedure applies to new installations of PDQuest software version 7 4 and higher or Quantity One software version 4 6 or higher To install the software 1 Open the Windows Control Panel click Start Control Panel and then open the Power Options dialog box 2 Set Turn off hard disks and System standby to Never If your computer goes into Standby mode it will cause EXQuest spot cutter to stop cutting 3 Close the Power Options and Control Panel dialogs 4 Insert the Discovery Series PDQuest or Quantity One installation CD in the CD ROM drive The disk will automatically start up with the Discovery Series installation window 5 Select the PDQuest or Quantity One Installer 6 Click Next on the Welcome Screen 2 8 SYSTEM INSTALLATION 7 Read and accept the License Agreement 8 Click Next on the
86. s Place the gels on the cutter at the specified positions relative to the stage center C White Trans When all the gels are on the cutter press Acquire image pAcauitre Image UVEPI Time secs Display Analysis image s showing gel placement Cancel Help Place the gel s or blot s on the gel tray and position them as shown in the image see Figures 6 10 and 6 11 for an example Make sure the gels or blots are held securely in place by the clamps provided with the spot cutter see Section 4 1 Failure to clamp the gel or blot may result in decreased accuracy and low cut yields Select the light source Use White Trans for visible stains such as Coomassie or silver stain or UV EPI for fluorescent stains such as SYPRO Ruby Click Acquire image to image the cutting deck Toggle between the Analysis image s showing placement and Image of cutter stage images to ensure that the gels have been placed on the platform in the correct location and orientation Click Proceed to continue to the Align Analysis and Cutter Images dialog box or Cancel to return to the Analysis Set Excision Control Tool window ANALYSIS SET EXCISION TOOL 14 Align the spots on the cutter image right side of Figure 6 12 with spots in the analysis set image left side of Figure 6 12 di Align Analysis and Cutter images sample vi Fhered a o D 4 Step 1 Place deta box Step 2 Patom aligrmert Step 3 Cordem g
87. s required to obtain an adequate amount of protein in each spot The tolerance specifies the range of quantities that are considered equivalent Any gel containing a spot within the range may be used Gels with spots above the range are preferred gels with spots below the range are rejected Tolerance r Ir 24 told above below average Include quality to help avoid spots that are highly overlapped i include quality in spot comparison The number of gels selected by the above criteria 2 gt 3 a g a Lo oe y 2 S o 3 Figure 6 7 Automatically Select Gels to Cut Screen ANALYSIS SET EXCISION TOOL From the Automatically Select Gels to Cut screen set the Tolerance The tolerance value is used to determine the spot quantity range that is acceptable when choosing the gel from which a particular spot will be cut If the quantity of a spot in only one gel falls within the tolerance that gel will be auto selected for the spot cut However if the quantity of a spot in two or more gels falls within the tolerance range then the auto select mechanism will consider which gels will best minimize the time and effort required to switch gels on the spot cutter platform For example if you have five gels in an analysis set with the tolerance ratio set to 1 the lowest setting auto selection specifies different cuts in all five gels requiring you to change gels in the spot cutter five times However by increasing the tolerance
88. set the water volume required to wash the gel tip This tab is not available in membrane cutting mode 8 8 RUN INFO PANEL Run Info Run Info Cuts 118 Min Well 72 Cuts 120 Min Well o ze See Volume e Volume Wells 108 Wells 108 ng E Well overflow Plates 2 Plates 2 Figure 8 8 Run Info panel The Run Info panel provides information about the cut run as it is set up Cuts This field displays the number of cuts requested and includes multiple cuts made from the same spot Wells This field displays the number of microplate wells required for the cut run This number will always be less than or equal to Cuts since multiple cuts made from one spot always go into the same well Plates This field displays the number of microplates required and depends on the plate format Section 8 5 and number of Wells Min Well Volume This field displays the minimum required well or tube volume Min Well Volume must be less than or equal to the ml well or ml tube volume selected in the Plate Options panel see Section 8 5 or a Well overflow warning will appear If a Well overflow warning is displayed either use a microplate with a larger well volume or reduce the Multiple cuts object value in the Cut Options panel see Section 8 4 EXCISION TOOL CONTROLS 8 9 PLATES FOR CUT RUN Plates for Cut Run Welt evolatie 288 BrcodeAd Check on wells to siap them dunng eraron eed leave them empty Y D
89. spot cutter are in an Error state Camera is not focused F stop set incorrectly Empty water bottle Air bubbles Loose tubing connectors Tip Damage Cutting sheet damage free gels Tip height adjustment Gel hydration Disable sleep mode on your computer see Section 2 9 Make sure that the power cord is connected and the power switch is turned on Close the software and turn the spot cutter off Turn the spot cutter back on wait for approximately 30 seconds and then restart the software Perform the camera focusing and camera calibration steps described in Section 3 Perform the camera focusing and camera calibration steps described in Section 3 The spot cutter relies on water aspiration for efficient gel pickup and deliver Check the water level and make sure that there is sufficient water Large air bubbles in the tubing reduce the gel pickup and delivery efficiency Start manual excision tool and click Prime Pump Prime the system until the air bubbles are removed Loose fittings cause air to get into the fluidics system Make sure all fittings are tight Damaged or bent tips do not cut gels efficiently Examine the cutting tip for damage and replace it if necessary Make sure that you are using the cutting sheet supplied with the spot cutter If it has been used several times or is severely scarred replace it See Section 9 Accessories for ordering information Do not remove the soft film from the
90. t However after you have imported a cut list you can then add additional cuts to the image To import a cut list 1 2 Set up a Manual Excision Tool cut run as described in Section 5 1 Steps 1 13 Click Import to open the Import Excision Coordinate List dialog box Ge O Ol 10 MANUAL EXCISION TOOL IN Import Excision Coordinate List X Skip first line of file Column a e E File Sample Import Data vs E Units mm cm inches Origin Upper left Lower left All cuts may be dragged at once with the mouse vd Done X Cancel LI Help Figure 5 5 Import excision coordinate list If the first line of your file is header information check Skip first line of file Enter the column number in which the coordinates are located For example if your file lists the spot number in column one the X coordinates in column two and Y coordinates in three then you would enter 2 for column X and 3 for column Y Columns must be separated by tabs or by columns Click the browse button to locate the coordinate file to import Select the appropriate coordinate measurement unit in the Units field Select the coordinate system origin Upper left or Lower left Once all the correct information has been supplied orange cut marks will appear on the gel image Click the mouse and drag all the cut marks as a group to better position them on the gel Click Done to accept the cut mark locations After you have
91. to determine from which gels spots should be cut from as well as to minimize the number of gels that need to be cut Go to Step 5 Automatic gel selection e Resume a cut run or edit a cut request list T Analysis Set Excision Wizard Analysis Set with Finished Cuts There are 204 finished cuts You may Create a new cut run of the same spots on dilerent gels Recut exating finished cuts gt 3 2 3 Si Go OD m A 2 2 O 3 Figure 6 4 Analysis Set with Unfinished Cuts 6 2 5 ANALYSIS SET EXCISION TOOL From the Analysis Set with Unfinished Cuts screen Figure 6 4 Select Resume cut run and finish these cuts and then click Next to finish cutting an already defined cut list Go to Section 6 2 Setting up a Cut Run Select Change gel assignments and begin new cut run and then click Next to modify the cut request list go to Create a cut request list above Note that changes cannot be made for spots that have already been cut Cut uncut gels or re cut previously cut gels T Analysis Set Excision Wizard Analysis Set with Finished Cuts There are 204 finished cuts You may Create a new cut run of the same spots on dieran gels Recut esting finished cuts gt 3 3 3 2 Go ke A u a 2 O 3 Figure 6 5 Analysis Set with finished Cuts From the Analysis Set with Finished Cuts screen Figure 6 5 Select Create a new cut run of the same spots on different ge
92. tter through the Manual Excision Tool Both software packages include the following functionalities Imaging editing tools including image acquisition Transform Crop Flip Rotate Text Tool and file handling tools such as Print Export to TIFF and Save PDQuest Basic and Quantity One Basic however do not include any image analysis capability The Manual Excision Tool is accessed through the Identify drop down menu in PDQuest or the Excision drop down menu in Quantity One For more details see Section 5 Gel and Blot Excision Spot Cutter Calibration Wizard The Spot Cutter Calibration Wizard is used to calibrate the spot cutter This wizard is accessed through the Edit Preferences drop down menu in PDQuest and Quantity One For more detail see Section 3 Spot Cutter Calibration Wizard Manual Excision Tool The Manual Excision Tool provides the functionality required to Image gels or blots with UV EPI or white Trans lighting Create a cut list Cut gels and blots Deliver protein spots to microplates for downstream automated processing and analysis Import cut lists from any 2 D software package where x y coordinates can be exported PDQuest Software The full featured PDQuest software provides additional capabilities that include image analysis automated spot picking database storage of raw 2 D electrophoresis gel data and the ability to acquire gel and blot images using any of several Bio Rad imaging systems These sca
93. unts of protein from a single spot and deposit it into a single well or tube Enter the maximum number of cuts that you want to allow in the Maximum cuts object field Select the microplate format to be used from the Plate Options panel see Section 8 5 and then specify the well volume and well loading order The well volume will determine the maximum number of spots that can be placed in a single well tube Review the Run Info panel see Section 8 8 for the number of cuts selected the number of plates required and the required plate well volume Press Proceed to finish creating the cut request list and to open the Plates for Cut Run dialog see Section 8 9 Enter the plate name and barcode plate ID in their respective fields The plate barcode can be entered using a barcode scanner or typed in To reserve wells on a microplate click the Edit button for the desired plate and then click on individual wells to reserve them Finish setting up the run as described in Section 6 2 6 17 LANE VOLUME EXCISION TOOL 7 0 LANE VOLUME EXCISION TOOL The Lane Volume Excision Tool is designed to provide a simple and efficient method for cutting lanes and bands from 1D gels and blots This tool is available only with the purchase of a full Quantity One software license 7 1 SETTING UP A CUT RUN 1 Make sure the spot cutter is set up as described in Sections 2 and 3 and that the required cutting head is connected to the spot cutter
94. vidual cells to select or deselect the cuts Note that this will automatically switch the selection mode to Select from list on the Setup tab To add a cut request click on a white cell to delete a cut request click on a blue cell To cut the same spot in different gels click on multiple white cells within the same row You can add as many cut requests in as many gels in the analysis set as you want When finished look at the Run Info panel and see if the changes that were made have resulted in a well overflow warning To clear the error use a microplate with a larger well volume reduce the Maximum Cuts Object value or deselect some of the duplicate cuts in the Review table here and below Review Table Controls Table options panel e Show quality Click Show quality to display the spot quality in parentheses following the spot quantity e Show ratio Click Show ratio to display a quantity ratio column e Show all gels Click Show all gels to display quantity data for all the gels not just for the gels selected to be cut Image Options Panel e Display Cuts Check this box to display the cut request circles in the image e Auto zoom on cuts Check this box to automatically zoom in on a spot when it is clicked in the Review table e Active gels only Check this box to display images only for those gels listed in the Review table 13 Identify the cutting tip size 1 0 mm or 1 5 mm in the Cut Options panel see Sectio
95. visualized if their data are mapped to the computer screen in a nonlinear fashion Adjust the Gamma slider handle to expand or compress the contrast range at the dark or light end of the range and to reflect this in the transform plot and preview window You can also type a specific value in the field to the right of the slider Calibrated Quantity When images have been acquired with a densitometer click Calibrated Quantity to display the image units in O D s EXCISION TOOL CONTROLS Image Panel Max This text box displays the intensity of the image s most intense pixel Image units are determined by the type of scanner used to create the image Min This text box displays the intensity of the image s least intense pixel Image units are determined by the type of scanner used to create the image Display Panel Invert Display Check Invert display to change the preview image from light spots bands on a dark background to dark spots bands on a light background and vice versa This function does not change the data just the display Highlight Saturated Pixels Check Highlight Saturated Pixels to color saturated pixels red This setting does not apply to Gaussian images Always auto scale Check the Always auto scale box to automatically perform auto scale each time the image is opened and to disable the High Low and Gamma slider controls Buttons Auto scale Click Auto scale to automatically optimize the selected image
96. well volume and loading order The well volume will determine the maximum number of spots that can be placed in a single well tube 11 Optional Check Hydrate in the Hydration Options panel to automatically hydrate the gel during the cut run Specify how often it is to be hydrated see Section 8 6 then click Define hydration area to identify the gel or blot location Gels and membranes should typically be hydrated at least once every hour 12 If gels are being cut enter the wash volume required to clean the gel cutting tip 50 to 500 ul see Section 8 7 in the Wash Options panel 7 2 CUTTING LANES Entire Lane mode is used to define the lanes from which cuts will be made 1 Select Entire lane in the Specify Cuts panel r Specify Cuts e Entire lane Vertical 3 100 Horizontal 10 Volume overlay 0 0 a 1 Show excisions on image Figure 7 2 Specify Cuts panel Entire lane selected 2 Click Frame Lanes m and then click in the image to create lanes in an adjustable frame In the pop up box that appears enter the number of lanes to create Check or uncheck Show excisions on image to show or hide cut request labels on the image 7 2 LANE VOLUME EXCISION TOOL e af em e EH up e DT ge ag E am T ED Se i i ben ei i f ar D ee ST mme ge we ap z

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