EXQuest™ Spot Cutter Instruction Manual Catalog numbers 165-7200 165-7201 EXQuest Spot Cutter EXQuest Spot Cutter With Computer On the Web at http://www.bio-rad.
TABLE OF CONTENTS TABLE OF CONTENTS SAFETY SECTION 1 INTRODUCTION ..................................................................................................................1-1 1.1 Overview................................................................................................................................1-1 1.2 System Description................................................................................................................1-2 1.2.1 Gel Imaging System ..................
TABLE OF CONTENTS SECTION 7 LANE VOLUME EXCISION TOOL........................................................................................7-1 7.1 Setting Up a Cut Run ............................................................................................................7-1 7.2 Cutting Lanes ........................................................................................................................7-2 7.3 Cutting Volumes ...................................................................
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 • 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.
INTRODUCTION 1.0 1.1 INTRODUCTION 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.
INTRODUCTION 1.2 SYSTEM DESCRIPTION The EXQuest spot cutter is a fully integrated system controlled through PDQuest™ and Quantity One® software. It 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 detailed description of each component is given below. UV EPI lights Robot Water and Waste Bottles Microplate Tray Wash Station Gel Tray and Transilluminator Controls and Status Lights Figure 1-2.
INTRODUCTION Figure 1-3. Light enclosure. 1.2.2 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 1.2.
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). • 1-4 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.
INTRODUCTION • Membrane Cutting Head. The membrane cutting head is an optional accessory that is used for cutting PVDF and nitrocellulose membranes. 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.
INTRODUCTION • Gel Tip Wash Station. The gel tip wash station is located on the left end of the wash station block. It 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.
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.
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.
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 cutter through the Manual Excision Tool.
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.
SYSTEM INSTALLATION ! 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.
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 the gel tray.
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.
SYSTEM INSTALLATION 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.
SYSTEM INSTALLATION 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 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 1. 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. If the membrane cutting head is installed, unplug its electrical connector from the PC board located on the robot arm. 4.
SYSTEM INSTALLATION 5. 2.8 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. 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 reservoirs. 1. Install the bottle holder. Open the door on the left side of the spot cutter.
SYSTEM INSTALLATION 7. Read and accept the License Agreement. 8. Click Next on the 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.
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.
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). Figure 3-3. “EXQuest Calibration Wizard” screen – uncalibrated.
EXQUEST CALIBRATION WIZARD Figure 3-4. “EXQuest Calibration Wizard” screen – calibrated. From this screen you may • • • 5. 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).
EXQUEST CALIBRATION WIZARD Figure 3-5. Camera focusing dialog. 6. 3-4 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. Remove the calibration target from the cutting deck if it is present. b. Place a gel cutting sheet in the gel tray and install the gel tray in the EXQuest Spot Cutter c. Check Lower head to adjust height to lower the cutting head to the gel tray. d.
EXQUEST CALIBRATION WIZARD 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. Figure 3-7. “Flat Field Calibration” screen.
EXQUEST CALIBRATION WIZARD 8. 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 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. Figure 3-8. “Lens Calibration” screen. 9. 3-6 System Position Calibration.
EXQUEST CALIBRATION WIZARD Figure 3-9. System position calibration. Manual Calibration a. If calibrating the membrane cutting head, install the plastic protective sleeve over the 1.0 mm cutting tip. b. 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. c.
EXQUEST CALIBRATION WIZARD Figure 3-10. Plate position calibration: plate selection. b. c. 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 in 0.2 mm 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 in the well. Figure 3-11.
EXQUEST CALIBRATION WIZARD d. Use the Raise/Lower slider to adjust the tip height. Typically, the gel tip should be placed about 0 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.
EXQUEST CALIBRATION WIZARD 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 be placed about 2 mm into its wash port and the Membrane cutting tip is about 2 mm or more into its wash port.
EXQUEST CALIBRATION WIZARD Figure 3-14. Calibration complete! screen.
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.
GEL AND BLOT PREPARATION 4.3 BLOT PREPARATION 1. Store PVDF and nitrocellulose blots in a container that keeps them protected and hydrated until needed for spot cutting. 2. 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. 3.
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.
MANUAL EXCISION TOOL Figure 5-2. Manual excision tool. 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.
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 µl, see Section 8.7) in the "Wash Options" panel. 14.
MANUAL EXCISION TOOL 17. 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. 18. Press Done to open the "Load Plate on Cutter" dialog box (see Section 8.10).
MANUAL EXCISION TOOL Figure 5-5. Import excision coordinate list. 3. If the first line of your file is header information, check Skip first line of file. 4. 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. 5.
MANUAL EXCISION TOOL Figure 5-6. Confirm cuts pop-up. 2. Read the instructions on the "Image Microtiter Plate" screen. Figure 5-7. “Image Microtiter Plate” window. 3. Without moving the gel, place a clean plastic cutting sheet on top of the gel. 4. 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. 5. Click Acquire image to obtain an image with the same exposure time as the cut request image. 6.
MANUAL EXCISION TOOL 5.3.2 Identifying Re-cuts from a Gel Image 1. When the cut run is finished, return to the "Manual Excision Tool" screen and click Confirm Cuts to acquire a confirmation image. 2. 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. 3. 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. 4.
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.
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: • Create a cut request list 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".
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.
ANALYSIS SET EXCISION TOOL 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.
ANALYSIS SET EXCISION TOOL a. 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.
ANALYSIS SET EXCISION TOOL • 9. 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.
ANALYSIS SET EXCISION TOOL 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.
ANALYSIS SET EXCISION TOOL Figure 6-10. Place Gel/Blot on Spot Cutter dialog box: Single Gel Placement Figure 6-11. Place Gel/Blot on Spot Cutter dialog box: Multiple Gel Placement 9. 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).
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). 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".
ANALYSIS SET EXCISION TOOL Figure 6-14. Align Analysis and Cutter Images: Step 2 – Perform Alignment Panel • Automatic Alignment. Automatic alignment uses a Gaussian fitting algorithm to align the cutter image spots with those in the analysis set image. If the 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.
ANALYSIS SET EXCISION TOOL 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 spots in the analysis set image with blue X's. These marks cannot be seen if Matches is checked. • Matches.
ANALYSIS SET EXCISION TOOL 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 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.
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 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.
ANALYSIS SET EXCISION TOOL 3. Check Analysis Set Excision Tool advanced user mode in the "Spot cutter" panel and then click OK. 4. 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. 5. Prepare the gel or blot as described in Section 4. 6. Make sure that the DI water bottle is full and the waste bottle is empty at the start of the run. 7. If cutting blots, fill three 1.
ANALYSIS SET EXCISION TOOL Figure 6-23. Step 2 – Select Gels: Select from List Auto-select a. Choose Auto-select to let PDQuest automatically select the best 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). Figure 6-24. Step 2 – Select Gels: Auto-select b. c. Set the Tolerance.
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. Figure 6-25.
ANALYSIS SET EXCISION TOOL 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.
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. 2. Prepare the gel/blot as described in Section 4. 3.
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 well volume and loading order.
LANE/VOLUME EXCISION TOOL Figure 7-3. Lane/Volume Excision Tool image with lanes 3. Click Stretch Frame 4. Optional. Make additional adjustments to the lane frame as needed. 5. and then click and drag the frame anchors to fit the frame over the gel. • Click Add/Adjust Anchors and then click on the top or bottom of the gel to add new anchors. Click and drag the lane anchors to adjust them.
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. 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. Figure 7-6.
LANE/VOLUME EXCISION TOOL Figure 7-7. Lane/Volume Excision Tool: Display Excisions 4. 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. 5. 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.
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 Figure 8-1. “Acquire Image” panel The “Acquire Image” panel is used to acquire gels and blot images. 1. Select a light source for image acquisition. • • 2. 8.
EXCISION TOOL CONTROLS 8.3 TRANSFORM DIALOG BOX The "Transform" dialog box is used to adjust the image brightness and contrast. It contains controls used to view the pixel intensity frequency distribution; manually or automatically transform the image; check for saturated pixels; or color the image. Transform Plot Preview Window Frequency Distribution Histogram Figure 8-3.
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.
EXCISION TOOL CONTROLS • 8.5 Material: Use this drop-down menu to select the gel type that will be cut, e.g., "Free gel", "Plasticbacked gel" or "Glass-backed gel". When the membrane head is installed, "PVDF" will automatically be selected. PLATE OPTIONS PANEL 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.
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.
EXCISION TOOL CONTROLS 8.9 PLATES FOR CUT RUN 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.).
EXCISION TOOL CONTROLS 8.10 LOAD PLATE ON CUTTER DIALOG Figure 8-10. “Load Plate on Cutter” dialog box This dialog helps the operator correctly place the microplates in the microplate rack. • 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. • 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.
ACCESSORIES 9.0 ACCESSORIES, SUPPLIES, REPLACEMENT PARTS Catalog Number Description 165-7208 165-7205 165-7209 165-7202 165-7203 165-7206 165-7204 165-7210 165-7215 170-3751 165-7206 165-7218 165-7212 165-7219 165-7217 EXQuest Cutting Sheets (S), pkg of 15 EXQuest Calibration Pucks (R), pkg of 10 EXQuest Gel Clamps (R), pkg of 4 EXQuest 1.0 mm Nominal Gel Cutting Tip (R) EXQuest 1.5 mm Nominal Gel Cutting Tip (R) EXQuest 1.
MAINTENANCE 10.0 MAINTENANCE 10.1 ROUTINE MAINTENANCE • 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. • 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.
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: 1. 2. 3. 4. Turn the power switch off and unplug the spot cutter from its power source.
MAINTENANCE Figure 10-4.
TROUBLESHOOTING 11.0 TROUBLESHOOTING Symptom Cause Solution Computer does not communicate with spot cutter or camera USB cable not connected Ensure that both the USB cable and power cord are connected Computer went into sleep mode Disable sleep mode on your computer (see Section 2.9) The spot cutter power switch has not been turned on. Make sure that the power cord is connected and the power switch is turned on.
TROUBLESHOOTING Symptom Cause Solution Hydration and clamping 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. Low membrane cutting efficiency Tip is dull, or damaged A membrane cutting tip that is dull or damaged will not cut PVDF and nitrocellulose membranes efficiently.
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.
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.
WARRANTY SYSTEM RECORDS Serial Number Date of Purchase Purchase Order Number Notes Service Record Date 12-2 Description of Problem Name Signature
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