Roboinject Manual
Information in this document is subject to change without notice. No part of this document may be reproduced or transmitted without the express written permission of Multi Channel Systems MCS GmbH. While every precaution has been taken in the preparation of this document, the publisher and the author assume no responsibility for errors or omissions, or for damages resulting from the use of information contained in this document or from the use of programs and source code that may accompany it.
Table of Contents Introduction About this Manual 1 1 Important Information and Instructions Important Safety Advice High Voltage Requirements for the Installation Compressed Air Supply Handling of the Carrier Handling of the Injector Mount ( = z-Axis) Handling of Injection Needles Regular Backups System File Modifications Guarantee and Liability Operator's Obligations 3 3 3 3 4 4 4 4 4 5 5 5 Roboinject Welcome to the Roboinject First Use of the Roboinject Installing the Roboinject Installing the Roboinj
Roboinject Manual iv Troubleshooting Troubleshooting Clogged Injection Needle Error Messages 67 67 68 70 Appendix Technical Support Service and Maintenance Cleaning the x/y Table Replacing the Plunger Contact Information Ordering Information About Preparation of Xenopus Oocytes Materials Chemicals Oocyte Removal Collagenase Digestion and Defolliculation Selecting Good Oocytes Plating Oocytes Technical Specifications 71 71 72 72 72 73 74 76 76 77 78 78 79 80 81 Index 83
1 Introduction 1.1 About this Manual This manual comprises all important information about the first installation of the Roboinject hardware and software and about the daily work with the Roboinject. It is assumed that you already have a basic understanding of technical and software terms. Thus, no special skills are required to read this manual. If you are using the Roboinject for the first time, please read the "Important Safety Advice" before installing the hardware and software.
2 Important Information and Instructions 2.1 Important Safety Advice Warning: Make sure to read the following advice prior to installations of the Roboinject. If you do not fulfill all requirements stated below, this may lead to malfunctions, breakage, or even fatal injuries. Obey always the rules of local regulations and laws. Only qualified personnel should be allowed to perform laboratory work. Work according to good laboratory practice to obtain best results and to minimize risks.
Roboinject Manual 2.1.3 Compressed Air Supply Even small amounts of water in the compressed air can lead to a corrosion of the carrier. Other contaminations can also result in malfunctions. Use only compressed air fulfilling the following standards. The air must be absolutely free of water, oil, and any particles. When air is compressed, the humidity in the air is concentrated into a smaller area, frequently condensing inside the air hoses. Use a water separator to remove water from the air.
Important Information and Instructions 2.1.8 System File Modifications 2.2 If you remove or edit text of an initialization ("ini") or configuration ("cfg") file, the software may cause severe problems. These files relate to hardware functions. A modification of these files may lead to malfunctions or even severe damage of the hardware. Always keep a copy of the original files. Only advanced users should modify program files like these.
3 Roboinject 3.1 Welcome to the Roboinject The Roboinject is a fully-automated all-in-one solution for injection of cDNA, mRNA and other compounds into Xenopus oocytes in standard 96 well plates. It is also suited for automated injection of other cells or organisms in standard (96, 384) as well as customized well plates. Main features: Compact and functional design, works fully computer controlled. Easy handling - neither special skills nor special equipment required.
Roboinject Manual Operation summary: Instead of having a vast amount of different pull down menus, there is one clearly structured menu bar and also a toolbar presenting commonly used commands. Therefore, you will quickly become familiar with the Roboinject. The first step is to set up your "virtual" well plate according to your "real" well plate. Enter a unique ID for the plate and / or assign a file name.
Roboinject 3.2 First Use of the Roboinject This section contains important information about the general concept of the Roboinject and the setup of the system. Please read the "Important Safety Advice" in the introduction prior to installing the Roboinject hardware and software. The Roboinject Hardware The Roboinject is compatible with standard lab equipment and can be easily integrated in your working environment. Software controls for all settings replace any knobs on the device.
Roboinject Manual The vertically moving injector mount (z-axis), holding the injection device, is designed specifically for the demand of high stability, speed and precision. It moves at a resolution of 20 μm; position and speed are software controlled. The injection device is composed of a linear motor driving a steel plunger which fits precisely in the provided injection capillaries.
Roboinject Injection and cultivation of Xenopus oocytes is performed using disposable standard 96 well plates, which are commercially available from several providers. Please see chapter "Ordering Information" in the Appendix. The oocytes are plated into the wells in a couple of minutes and can be kept for several days. They quickly settle within the cone-shaped wells and adhere to the well bottom after a few hours.
Roboinject Manual 3.3 Installing the Roboinject Setting Up the Roboinject 1. Place the Roboinject on a rigid, stable, and vibration-free base in an air conditioned room. 2. Provide a power supply in the immediate vicinity of the installation site. 3. Provide a compressed air supply in the immediate vicinity of the installation site. 4. Place the computer and its accessories next to it. Connecting the Roboinject The following illustration shows the rear panel of the Roboinject.
Roboinject Connecting the Roboinject to the pressure line 1. Connect one end of the provided compressed air hose (ID 2.5 mm, OD 4 mm) to the water separator. 2. Connect the other end to the compressed air inlet on the Roboinject's rear panel, labelled with "AIR IN". 3. Connect the other provided compressed air hose (ID 4 mm, OD 6 mm) to the provided water separator. 4. Make sure that the pressure line is closed.
Roboinject Manual 3.4 Installing the Roboinject Software System requirements Software: One of the following Microsoft Windows ® operating systems is required: Microsoft Windows 7, Vista or XP (English versions supported with the NT file system). Other language versions may lead to software errors. Hardware: An USB 2.0 High Speed serial bus must be provided for the communication between the Roboinject and the computer. Additional 1 GB RAM working memory is strongly recommended.
Roboinject 3.5 Roboinject Software Starting the Roboinject Press the main switch on the back of the Roboinject for switching on the Roboinject. The Roboinject is starting now. After a few seconds the Roboinject is ready for use. Starting the Roboinject Software You may use the computer and the software offline that is, the Roboinject is switched off or not connected to the computer, to review already injected plates or define new injection protocols.
Roboinject Manual The Roboinject main window opens.
Roboinject 3.6 Main Menu For maximum flexibility all relevant parameters for the injection can be changed by the user via main menu. Note: The preset default values have been extensively tested with RNA and DNA injection of Xenopus oocytes and should work in most circumstances. Therefore, it is advisable to use the default values for the first injections with Xenopus oocytes. File Menu for creating new files, opening existing files, and for saving them.
Roboinject Manual Click "New" in "File" menu. The following "Plate Info" dialog opens. Please give an appropriate file name in "Plate ID". The type of the well plate can be defined from the "Plate Type" drop down menu. If you want to set the selected plate type as default, please enable the check box "Set As Default". You can type in notes concerning the actual experiment into the "Comment" text box. All information will be stored in the plate info (*.rpf) file, and displayed in the report sheet.
Roboinject Click "Open" in "File" menu. The "Open" dialog box appears. Browse your folders and select the Roboinject plate info file (*.rpf) you want to open. Click "Open". The main window now shows the state of the already saved plate. Click "Save" to save a Roboinject plate info (*.rpf) file. It will be stored in the selected "Plate Info" directory. Click "Save Template" and "Open Template" to open and save a template (*.rit) file. It will be stored in the selected "Template" directory, respectively.
Roboinject Manual Settings Menu for setting options and for defining directories. Click "Options" in main menu "Settings". The "Options" dialog has four tabs. Click "General" tab. The dialog allows general settings concerning two general software features, and the "Test Injection Dialog" in particular. Enable the check box "Auto increment plate ID", if you want that the plate ID is automatically counted up when creating a new file.
Roboinject Click "Aspirate" tab. This dialog allows to define the aspiration of compound from the reservoir tubes. The parameters are the “Velocity” of aspiration in nl/s, the “Surplus Volume” in nl, and the “Pause after Aspirate” in ms. “Velocity”: The aspiration velocity for a given compound depends on its viscosity. Samples with high viscosity should be aspirated slower than samples with low viscosity.
Roboinject Manual Click "Inject" tab. The dialog allows to define parameters concerning the “Injector Mount” (z-axis) and the “Injector” device itself. The settings are attributed to the different "Sample Types" such as RNA, DNA or others. “Sample Type”: Define the type of compound to which you want to assign the setting parameters from the drop down menu. “Injector Mount = z- Axis”: You can separate the impalement process of the injection needle into an oocyte or another cell in two phases.
Roboinject Example on the picture: The bottom of the well is value zero, the reference value. If you define, for example an impalement position of 650 μm, the injector mount will move down until its position is 650 μm above the bottom of the well. If you additionally define an injection position of 550 μm, the injector mount will move down again until its position is 550 μm above the bottom of the well.
Roboinject Manual Adjusting the Injection Parameters for each Well separate It is possible to adjust the settings for the injection for each single well separate. Click with the right mouse button onto a well in the virtual well plate, and the following dialog appears. Now you can adjust the injection parameters for this specific well and for each sample separate.
Roboinject Click "Rinse" tab. Rinsing the injection needle between the injection of different samples is mandatory for preventing cross-contamination. In principal, you can reuse an injection needle as long as it is not clogged or broken. Nevertheless, MCS does not recommend to reuse the needle longer than one day after the last injection.
Roboinject Manual Tools Menu for manual mode and for creating a report sheet. The option "Set Plate to Manually Injected" offers the possibility to document information about a well plate, loaded with manually injected oocytes which will be processed with the Roboocyte1 device. Please see below. About software controlled manual manipulation of the Roboinject, please read the next chapter "Toolbar" "Open Manual Mode Dialog". Click "Show Report".
Roboinject Click "Set Plate to Manually Injected". When you are using the Roboocyte1 device from Multi Channel System MCS GmbH and you decide to inject manually, or if you performed manual injection with Roboinject for other reasons, you can define the oocytes of a well plate as "injected" with this function. Click "File" menu “New” to create a new plate. Fill in the respective information about the well plate, created and injected in Roboocyte1.
Roboinject Manual Help The menu offers the use of the Roboinject online help, and shows the "About Roboinject" dialog. Click “Roboinject Help” to start the online help. Click "About". The dialog displays the actual used version of Roboinject software, and hardware information.
Roboinject 3.7 Toolbar Click the "New File" icon . The "Plate Info" dialog opens. Please read the description in chapter "File" of the main menu. Click the "Open Manual Mode Dialog" icon .
Roboinject Manual The "Manual Mode" dialog opens. The dialog is divided in five sections: Global, Plate Carrier, Air Pressure, Injector Mount and Injector. That means, you are able to influence each part of the device manually via software controls. The commands inside the sections refer to the plate carrier, the injector mount (= z-axis) and the injector (= plunger) only. The "General" section refers to the complete device.
Roboinject Plate Carrier Section Click "Reference". The carrier performs its reference movement in x/y direction. If the carrier does not move, please check all cable connections. Warning: Perform the alignment procedure before using any of the following commands. Please double-check whether the z-axis is in "Home" position whenever carrier movements are elicited. Otherwise there is the risk of severe damage to the injector. Click "Home" for moving the carrier in home position.
Roboinject Manual Air Pressure Section Check the air pressure. It should be set to a minimum value of 3000 hPA, and should never exceed a value of 4 bar. Injector Mount = z-Axis Section Click "Reference". The z-axis will perform its reference movement and will stop in home position. The depicted Z value is 0 μm. Click "Home" to move the z-axis into home position. The home position for the z-axis is defined as the position when the injection device is at its topmost position. The depicted Z value is 0 μm.
Roboinject Injector = Plunger Section Click "Reference". The plunger will perform its reference movement and will stop in home position. Click "Home" to move the injection plunger inside the z-axis into home position. The home position is defined as the topmost position. Click "Replace" to start the needle installation or needle changing. Click "Empty" to move the injector plunger continuously from the topmost position into the injection needle to extrude the oil filling of a new injection needle.
Roboinject Manual Alignment of the Well Plate Carrier The well plate carrier hovers over the magnetic x/y table. For a smooth and proper movement, it is crucial that the table is kept dry and clean, and the carrier is properly aligned on the table. Clean the x/y table with a soft tissue and pure alcohol. Click on the icon to open the "Manual Mode" dialog. Activate the air cushion by clicking on the "Air Cushion" button. Now it is possible to move the carrier carefully by hand.
Roboinject 3.8 Control View The "Control View" on the right side of the main window of the Roboinject software is divided into two smaller windows: "Injection" and "Log" file. In the "Injection" window the status LED indicates the status of the device. Green color indicates a valid connection between Roboinject and computer, red color indicates no connection. The "Start" and "Break" buttons start and interrupt the injection process.
Roboinject Manual "Injection" Window After completing the definition of the injection protocol the "Start Injection Wizard" proceeds with the final preparations for the injection. Via "Start" and "Break" buttons you start and stop the injection manually. The status bar under the buttons will show the processing of the injection. Click the "Start" button. The "Preparation" dialog of the "Start Injection Wizard" appears. Air pressure: Please check the air pressure, the minimum has to be 3 bar = 3000 hPa.
Roboinject Click "Next". The "Alignment" dialog of the "Start Injection Wizard" appears. Plate Carrier: It is possible to move the plate carrier stepwise with the arrow buttons in x- and y- direction on the table. The actual position of the carrier in relation to the home position (X = 0 μm, Y = 0 μm) is displayed. Use the arrow buttons to move the carrier stepwise in 30 μm steps (slow) or continuously (fast) to any desired position.
Roboinject Manual The "Test Injection" dialog appears immediately before starting the injection process of the selected sample. This dialog gives the user the possibility to check the transmissibility of the tip opening of the injection needle. The defined sample volume will be injected into air. Before doing so, please place the microscope on the x/y table in a way that you can clearly see the needle tip.
Roboinject To control the ongoing injection process you can choose between three possibilities: Click "Continue" to continue with injection process. Click "Cancel" to stop the injection process. The carrier and the injection mount will automatically move to home position. Click "Stop" to stop the injection process without any movement of carrier or z-axis. This is useful in case of severe malfunctions which could possibly lead to damages if further movements would be performed.
Roboinject Manual 3.9 Tube View In the "Tube View" you control the definition and selection of the samples. You can use the eight reservoir tubes for eight different samples. Click onto the symbol of a "Edit Sample" icon. The "Sample Information" dialog opens. This dialog allows the definition of the respective injection samples inside of the 0.5 ml micro reaction tubes. Note: The sequence of injection is always from left sample (No 1) to right sample (No 8).
Roboinject Click the "Set Injection" button for setting injection parameters for that sample. Ignore the button for keeping the default settings. Click "OK" to complete the settings for this sample. Click "Clear" if you want to restart with default values, and "Cancel" if you want to go back to the main window without changes. Repeat the settings in "Sample Information" of other tubes until all samples you want to inject are defined.
Roboinject Manual 3.10 Well Plate View This prominent "Well Plate View" shows a 96 well plate. When using a 384 well plate, this view is automatically adapted. . To select or deselect a single well, please click onto the respective Click the "Void Well" icon well. To select or deselect complete rows or columns, click onto a letter on the left side to mark a horizontal row, or onto a digit on top to mark a column. Deselected wells are crossed out, and will not be injected.
Roboinject Click the "Clear Plate" icon to set back all wells to the original, non-selected status. The deselected (crossed out) wells will remain.
Roboinject Manual Defining the Samples After setting up the "Sample Information” dialog, the samples of the tubes are fixed. To define which well should be injected with which sample, please click onto the tube icon first, and then onto the well icon. The color of the well will immediately change into the symbol color of the sample of the selected tube. Choose another tube to assign one or several wells to that tube sample respectively. It is possible to assign any well to any sample.
Roboinject Defining the Number of Injections into a single Oocyte In some experiments it is necessary to inject more than one compound into a single oocyte. The Roboinject program gives the possibility for up to four injections into the same oocyte. To define the well for several injections, please click onto the tube icon first, and after that onto the well. The color of the well will immediately change into the symbolic color of the sample of the selected tube.
4 Injection 4.1 About Injection with the Roboinject Manual injection is often a limiting factor in high-throughput screens. The Roboinject injects Xenopus oocytes and other large cells or small embryos in a fully automated way. For example, injection and cultivation of Xenopus oocytes are performed using disposable standard 96 well plates, which are commercially available from several providers. The oocytes are plated into the wells in a couple of minutes and can be kept there for several days.
Roboinject Manual 4.2 Strategies for a Successful Injection Multi Channel Systems provides injection pipettes with an inner diameter exactly matching the plunger wire. The provided injection pipettes are glued to luer-fittings and therefore ready to use. If you nevertheless decide to fabricate your own injection pipettes you should keep in mind that you can only use the capillaries listed in the chapter "Ordering Information" in the Appendix. Other capillaries will not work properly.
Injection Xenopus oocytes The impalement and injection distances for cDNA are preset to 300 μm and 550 μm, respectively. (from the bottom of the well = 0 value). These distances have been shown as the optimum values for DNA injection. The overall expression rate you can achieve by this method is 80 percent of the oocytes. You can define your own impalement and injection depth, but MCS recommends using the default values in order to achieve the best expression rate.
Roboinject Manual 4.3 Preparations for Injection Generally, you can use cDNA or mRNA concentrations and injection volumes as described in the literature or as applicable for your experiment. In the following, typical cDNA or mRNA concentrations and recommended treatment of the sample are described. Please follow this advice if you are new in operating the Roboinject or if you have observed trouble with the injection process.
Injection Preparing the sample for injection 1. Take the sample out of the freezer at least 10 minutes before the injection. 2. Let it stand at room temperature until the sample is completely thawed. 3. Mix the sample thoroughly, for example, by vortexing for a short moment, or manually by striking the side of the tube with a finger. 4.
Roboinject Manual 4.4 Setting Up the Well Plate 4.4.1 Inserting or Changing the Well Plate Click the icon to open the “Manual Mode” dialog. Select “Change Plate” in the “Global” section to move the carrier into home position. Remove the used well plate if necessary. Place the type of well plate which is defined in the “Plate Info” dialog in correct orientation on the carrier.
Injection Creating a new file either on the toolbar or on the "File" menu. 1. Click "New File" icon The "Plate Info" dialog box opens. 2. Type in the "Plate ID", select the "Plate Type", and all information ("Comment") you want to associate with this well plate, for example the oocyte batch, name of the operator, and so on. 3. Click "OK" when you are finished. The "Save As" dialog box is displayed. The plate ID is also the suggested file name. 4.
Roboinject Manual Opening a previously saved file 1. If you want to inject the same plate again or review previously injected cells, open the appropriate well plate file. 2. Click "Open" on the "File" menu. The "Open" dialog box appears. 3. Select the Roboinject file (*.rpf) you want to open. 4. Click "Open". The well plate window now shows the status of the already saved plate; and the Roboinject header shows the name of the actually opened file. Defining the Injection Samples 1.
Injection Enter an ID and / or a name which univocal identifies your sample. Select the type of “Sample” and its “Concentration”. Note: The minimal volume which always has to remain in the tube reservoir is 1 μl. For example, if you need 4 μl sample volume for your injection run, please make sure that you start with at least 5 μl. This is because the injection needle keeps always a safety distance to the tube bottom to avoid uptake of contaminants and plugging.
Roboinject Manual Enable the check box "Test Injection" if you want to use this feature. Write a comment in the "Comment" text box. Click "OK" if you finished the settings of the sample, "Clear" if you want to restart with default values and "Cancel" if you want to go back to the main window without changes. Repeat the steps above until all samples you want to inject are defined.
Injection 4.5 Selecting Oocytes for Injection The next step after definition of the injection samples is the allocation of the sample(s) to the respective wells. Click the "Void Well" icon to exclude certain wells from injection. The deselected wells will be crossed out. Click the "Clear" icon non-selected status. to set back the virtual well plate to the origin, First, please select an injection sample.
Roboinject Manual 4.6 Changing the Well Plate and Loading the Needle You will need the cDNA or mRNA samples, the well plate loaded with oocytes, and an injection needle. You can reuse injection needles several times as long as they are not clogged or damaged. You will also need the provided stereo microscope and the provided alignment device (with crosshairs) in order to position the well plate horizontally and vertically as well as the injection needle before you start the injection.
Injection Changing or loading the injection needle 1. Use the "Change Needle" command to change or install a new needle. "Change Needle" will move the plunger and carrier in home position and move the z-axis a bit down for better access to the luer connector for the needle. 2. Use a fine pipette tip, for example, a microloader filled with mineral oil. Insert the fine tip into the needle as close as possible to the needle tip. Fill the needle completely and air-bubble free with oil.
Roboinject Manual 4.7 Alignment You have to adjust the position of the carrier and the needle under microscopic control. The Roboinject always moves relative to a standard = home position. The aim of the alignment process is to define this standard position. If this is not done properly, the Roboinject moves to wrong positions. The alignment process also compensates for minor changes in the dimensions of different well plates and variations in the length of the needle.
Injection Open the “Alignment” dialog in the “Start Injection Wizard”. 1. Switch in "Plate Carrier" to "Fast". The movement of the z-axis and the carrier commanded by the software controls will now be fast. 2. Move the injector mount (z-axis) and the carrier by pressing the arrow keys on the alignment dialog until the needle is almost centered on the alignment device. 3. Switch to "Slow". The movement will now be carried out in small single steps. 4.
Roboinject Manual 5. Click ”Finish” after successful alignment. 6. The current position is saved, and the old alignment position is overwritten. All following movements relate to the new standard position now. Warning: Do not click "Next" if the alignment has not been finished. This could lead to wrong movements of the Roboinject, which are potentially hazardous. If you want to cancel the alignment, leave this dialog box by clicking "Cancel" or "Back".
Injection 4.8 Starting the Injection To start the injection after successful alignment, simply click the "Finish" button in the Alignment dialog window. After the oil has been pushed out to the waste tube and the volume of the first sample has been aspirated, the "Test Injection" dialog appears if activated. This dialog gives the user the possibility to check the transmissibility of the tip opening of the injection needle. The respective sample volume will be injected into air.
Roboinject Manual The "Injection Halt" dialog appears. Click "Break" to stop the test injection and the general injection process, for example, if the injection needle is broken or clocked. To control the ongoing injection process you have three possibilities: Click "Continue" to continue with injection process. Click "Cancel" to stop the injection process. The carrier and the injection mount will automatically move to home position.
Injection 4.9 Saving the Well Plate After a successful injection process of all samples the Roboinject software displays the "Injection Finished" dialog. Click "OK" to continue with the same well plate or remove the plate and click "OK". The "Plate Info" file including the well plate status, file info, and comments is saved automatically to the destination you have specified in main menu "Settings", "Directories".
5 Troubleshooting 5.1 Troubleshooting Most problems occur seldom and only under specific circumstances. In most cases, it is only a minor problem that can be easily avoided or solved. If the problem persists, please contact your local retailer. The highly qualified staff will be glad to help you. Please inform your local retailer as well, if other problems that are not mentioned in this documentation occur, even if you have solved the problem on your own.
Roboinject Manual 5.2 Clogged Injection Needle If you observe that your injection needles are frequently blocked, please check the blocked needle under a microscope to find the cause of the problem. Injection needle is blocked from outside DNA / RNA solution flows out of the tip and dries out. The sticky residues will block the tip.
Troubleshooting Injection needle gets blocked during injection If the injection needle is fine when you start the injection, but gets clogged during the injection sequence, impurities in the DNA / RNA sample can still be the cause, because the particles may sink down slowly. Check the possible causes for the problem "Injection needle is blocked from inside" as well as the following. Possible causes: ? Contaminations in the cDNA / mRNA sample slowly sink down during injection and cause the needle to clog.
Roboinject Manual 5.3 Error Messages The alignment was not properly performed. Please repeat the alignment procedure. The plate file is not compatible. If you rename a plate file, please do not use the following signs.
6 Appendix 6.1 Technical Support Please read the "Troubleshooting" part of the manual or help first. Most problems are caused by minor handling errors. Contact your local retailer immediately if the cause of the trouble remains unclear. Please understand that information on your hardware and software configuration is necessary to analyze and finally solve the problem you encounter.
Roboinject Manual 6.2 Service and Maintenance Please make sure that you read this chapter thoroughly. Proper handling and cleaning of the Roboinject will ensure reliable and long life time of the equipment. Most often, troubles are simply due to mishandling. See also chapter "Troubleshooting". 6.2.1 Cleaning the x/y Table You prevent a malfunction of the carrier by cleaning the x/y table regularly. Use a soft tissue and pure alcohol (100 %) to clean the x/y table.
Appendix 6.3 Contact Information Local retailer Please see the list of official MCS distributors on the MCS web site. User forum The Multi Channel Systems User Forum provides the opportunity for you to exchange your experience or thoughts with other users worldwide. Mailing list If you have subscribed to the Mailing List, you will be automatically informed about new software releases, upcoming events, and other news on the product line.
Roboinject Manual 6.4 Ordering Information Sources of Supply Multi Channel Systems MCS GmbH recommends the use of the products tested with the Roboinject system. Well Plates Product Product Number Description Supplier V96 MicroWellTM Plates 249570 Clear polystyrene, conical, non-treated, non-sterile Nunc www.nuncbrand.
Appendix Oocyte preparation Product Product Number Description Supplier Xenopus oocytes Freshly prepared and ready-to-use EcoCyte Ecocyte Bioscience www.ecocyte.de Oocyte filter For selecting oocytes of appropriate size Multi Channel Systems MCS GmbH www.multichannelsystems.
Roboinject Manual 6.5 About Preparation of Xenopus Oocytes The materials and methods described in the following are kindly provided by the Bayer AG, Leverkusen. The described procedures have been optimized over several years for best performance of the oocytes and highest throughput. We recommend that you follow the instructions for obtaining best results. Refer to standard protocols if you need more detailed information on the subject.
Appendix Rubber ball for oocyte transfer pipette Glass tool for moving oocytes: Glass Pasteur pipette, sealed over a Bunsen burner, and melted into a golf-club like shape. Petri or cell culture dishes, 100 mm Petri dishes, 60 mm Beaker, 100 ml Razor blade Forceps Parafilm Standard laboratory equipment 6.5.2 Chemicals Collagenase For defolliculation: Fresh 1.52 mg/ml collagenase in Barth’s solution without Calcium.
Roboinject Manual Barth’s solution without Ca2+ pH 7.4 (with NaOH) 88 mM NaCl 2.4 mM NaHCO 1 mM KCl 0.82 mM MgSO * 7 H O 5 mM Tris/HCl 3 4 2 6.5.3 Oocyte Removal 1. Remove the appropriate amount of ovarian tissue surgically from one side of the frog. Please refer to standard protocols on this subject (Stühmer and Parekh 1995). 2. Transfer the ovarian lobes into a new large Petri or cell culture dish (for example 100 mm Falcon) filled with Barth’s without Ca2+. 6.5.
Appendix 6.5.5 Selecting Good Oocytes Rough selection by filtration 1. Fill a 100 ml beaker with 80 ml of Barth's solution. Place the oocyte mesh sieve into the beaker for grading the oocytes by size. The mesh of the sieve should be completely immersed in the fluid. 2. Pipette an amount of oocytes onto the sieve. Approximately half the sieve should be covered with oocytes. Too many oocytes on the filter will lead to an inefficient filtration. 3.
Roboinject Manual 6.5.6 Plating Oocytes You need a well plate filled with Barth's + gentamicin. Important: Visually check all well plates before use. Use only unsterile and very flat plates. Sterilization may lead to warped plates. Warped plates will decrease the injection and expression efficiency. 1. Aspirate a number of oocytes with a transfer pipette by applying a low negative pressure, for example, with a rubber ball. 2.
Appendix 6.6 Technical Specifications General information Operating temperature 10 °C to 50 °C Storage temperature 0 °C to 70 °C Dimensions (W x D x H) 320 x 320 x 310 mm Weight 23 kg Power 30 W Power Supply Unit FSP 150 - ABA AC Input 100 V to 240 V ~ 2 A @ 50 - 60 Hz DC Output 24 V = 6.
7 Index 83
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A H Air Cushion 13 Air pressure 13 Alignment 13, 56 Start Injection Wizard.......................56 Alignment Device.......................................56 Alignment Dialog.................................13, 56 Alignment of the Carrier 13 C Changing the Injection Needle 13 Changing Well Plate 54 Coarse Position..................................54 Installing Needle ...............................54 Loading Needle .................................54 Start Injection Wizard.................
Roboinject Manual Selecting good Oocytes ....................71 Preparations for Injection 47 Preparing a Sample...........................47 Quality of Oocytes ............................47 RNA / DNA Concentration ................47 R Reference 13 Replacing the plunger 65 Roboinject 1 Running a Test Injection with a Dye 45 Trypan Blue .......................................45 Manual Mode............................................. 13 New File......................................................
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