d. Bands hidden by overexposure with chemiluminescence become clear when imaged with Odyssey.

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1 Core Equipment ID: Description: LI COR Biosciences, Odyssey Infrared Imaging System Room: B446 (Molecular & Biochemical Core) Champion: 1.0 Purpose Standardize the process for control, maintenance, ownership and use of the LI COR Biosciences Odyssey Infrared Imaging System located in B 446 Life Sciences. 1.1 LI COR Biosciences, Odyssey Infrared Imaging System Capabilities 1 The Odyssey Infrared Imaging System sweeps away the old paradigms of chemiluminescence and visible fluorescence, and introduces a new standard for Western blot analysis direct infrared fluorescence detection. a. Infrared detection gives you the quantitative analysis and wide linear dynamic range that chemiluminescence can t. b. Strong and weak bands on the same blot are accurately detected without the uncertainty and inconvenience of multiple exposures, and without spending time in the darkroom. c. The Odyssey System gives you clear, sharp, reproducible bands without fuzziness or blowout. d. Bands hidden by overexposure with chemiluminescence become clear when imaged with Odyssey. LI COR Biosciences, Odyssey Additional Capabilities 2 The Odyssey System provides a flexible, multifunctional platform to accommodate a variety of applications, so you get results faster. a. Western Detection including two color blots and in gel westerns 1 LI COR Biosciences Biotechnology Product Line. LI COR Biosciences. April 1, LI COR Biosciences Odyssey: Clear Image Data. LI COR Biosciences. April 1, Author: Janice Thompson Page 1 of 9 Approval Date:

2 b. Cell Based Assays including in cell westerns and on cell westerns c. Small Animal Imaging d. Protein Detection including 1 D/2 D Coomassie Arrays e. Nucleic Acids including EMSA/Gel Shift and DNA Staining f. Microwell Assays including ELISA/FLISA, transcription factors and arrays g. Tissue and Tissue Sections 2.0 Reason for Issue Maintain a document that describes the Standard Operating Procedures that allows for the standard safe and maximal use of the LI COR Biosystems Odyssey within the Pharmacology and Toxicology Core Facilities. 3.0 Process Description Allows Core Facility Users within the Pharmacology and Toxicology Department to properly and effectively use the LI COR Biosciences Odyssey. The process description details and creates a standard for the use of the LI COR Biosciences Odyssey. The controlled standard must maintain and adhere to proper and approved research and regulatory qualitative conditions. 3.1 SOP: B for LI COR Biosciences Odyssey, authored by Janice Thompson, created on April 1, 2009, issued on (insert date). 3.2 SOP: B applies to any user and/or trainer of the LI COR Biosciences Odyssey. 3.3 Responsibilities: All Users are responsible for obtaining the proper approval and training before the use of the LI COR Biosciences Odyssey. All users are responsible for the proper use, according to defined protocol, when using the LI COR Biosciences Odyssey. a. New Users need a new LI COR Biosciences Odyssey user name and password created for equipment access, before initial use. New accounts are authorized and created by the Equipment Champion and/or the Core Facility Manager. A new account may be created after training and equipment approval has occurred. Author: Janice Thompson Page 2 of 9 Approval Date:

3 b. All Users must schedule equipment use in the Equipment Schedule Logbook prior to use. The Equipment Schedule Logbook is located on the desk next to the LI COR Odyssey. Please note that the Nanodrop Technologies Nanodrop ND 1000 Spectrophotometer is run on the same computer as the LI COR Biosciences Odyssey and Users must check the Equipment Schedule Logbook for this piece of equipment when considering the use of the LI COR Biosciences Odyssey. c. All users must record all equipment use in the Equipment Usage Logbook post use on the same day as the recorded use. The Logbook is located on the desk next to the LI COR Biosciences Odyssey. Within the Logbook on the current log sheet, Users must record the following: Date, PI, Name, Lab Location, Phone Number, Channel(s) Used, Number of Scans, Comments. d. Immediately after use, the LI COR Biosciences Odyssey must be cleaned with distilled water and a Kimwipe to ensure residue does not remain on the scanning surface. 3.4 Equipment Safety Issues a. Laser 3 The Odyssey Infrared Imaging System contains two lasers; one emitting at 785 nm, and one at 685 nm. Each has a peak power rating of 30 milliwatts. The 685 nm laser emits visible laser radiation direct exposure to either beam may cause eye damage. Laser radiation is emitted through apertures at the top of the Odyssey microscope assembly. Because the microscope assembly moves in both planes relative to the glass scanning surface, laser radiation could be focused at any position on this surface. Safety interlocks automatically turn the lasers off when the Odyssey lid is opened. b. Safety Interlocks 3 The Odyssey Infrared Imager has two safety interlocks that prevent access to the laser when the instrument lid is opened during operation. Do not attempt to defeat these interlocks. The interlocks are located on the front of the instrument bezel as shown in the Operator s Manual, Version 2.1. Audible tones sound when the lid is opened and closed. c. Chemical Safety 3 LI COR recommends that all biochemicals be handled carefully, and that safe laboratory procedures be followed 3 Odyssey Infrared Imaging System Operator s Manual, Version 2.1. LI COR Biosciences. Author: Janice Thompson Page 3 of 9 Approval Date:

4 at all times. Be aware of the hazards associated with any chemical before you begin work. d. Radiation Safety 4 The Odyssey Imager should not be used with any radioactive materials. 3.5 Quality Measures a. Each Use: Each time the LI COR Bioscience Odyssey is used, wipe all chemical spills and residues from both the case and the scanning surface. This prevents the surface coating from being damaged. Use only warm water and a damp cloth to clean the LI COR Biosciences Odyssey. b. Every Six Months: Inspect cables and power cords to ensure their integrity. 3.6 Procedure for Western Blot Scanning; LI COR Biosciences Setup a. Double click on Odyssey V3.0 software desktop icon to start use. This software runs the LI COR Biosciences Odyssey system. b. When initial screen has loaded, click on File and select New from the dropdown menu. c. In the prompt box that opens, select Browse from the right side of the box. Within the window that opens, select the folder to save the file in. Ensure that it is within the folder for your laboratory only. d. In the File Name area, type in the name of the file. e. Select Scan from the bottom of the window. f. When the Please note the project location message appears, click OK. g. Type in your User Name and Password. (These will be assigned to you when training has been completed.) Click OK. h. Open the lid of the LI COR Biosciences Odyssey. A soft, short beeping sound will be heard. This indicates the laser has been 4 Odyssey Infrared Imaging System Operator s Manual, Version 2.1. LI COR Biosciences. Author: Janice Thompson Page 4 of 9 Approval Date:

5 turned off and that it is safe for the User to work on the scanning surface. i. Place the membrane face down on the lower left corner of the scanning surface, noting the coordinates from the scanning surface. Roll the membrane with the brayer to remove air bubbles. j. Close the lid of the LI COR Biosciences Odyssey. A soft, short beeping sound will be heard. This indicates the safety interlocks will allow the laser to function when the parameters have been set. k. Identify Group from the dropdown menu. This should be the same as your User Name. l. Identify the Preset. For western blots, choose Membrane. m. The Scan Parameters remain at the factory default settings. n. Identify the wavelength to be scanned. This will coincide with the label attached to the secondary antibody. If using both channels, check boxes for both the 700 and 800 nm wavelengths. o. The Intensity remains at the factory default settings. p. Identify the scan area by typing in the coordinates OR resizing the highlighted red box on the grid. To resize the red box, hover over a line with the cursor until it changes from a four sided arrow to a two sided arrow. q. Select Start Scan. The LI COR Biosciences Odyssey will connect with the computer and the scanning process will begin. A noise will be heard from the Odyssey while the laser is moved back and forth over the area to be scanned. As lines are transferred to the computer, an image will appear in the highlighted area on the grid. r. When scan is complete, press Save. Type in the scan name and analysis name, using features identifiable for future reference. Press OK. s. Close the Scanner Console screen by pressing the red X in the upper right corner or press the Close button in the lower right corner of the screen. Analysis a. Click on View and select Alter Image Display from the dropdown menu. Author: Janice Thompson Page 5 of 9 Approval Date:

6 b. Adjust Brightness and Contrast levels for the appropriate channels by sliding the blue square on each bar until the desired levels have been achieved. c. To further enhance the image, the Linear Manual can be changed by sliding the blue square on this bar. Further adjustments to the Brightness and Contrast levels can be made after the Linear Manual number has been changed. d. Black and White can only be used as an option when a single channel is selected. e. When finished, press OK. f. If everything is acceptable, click on File and select Save from the dropdown menu. g. The image can be printed (click on File and select Print from the dropdown menu, then follow the prompts) or saved to a jumpdrive and taken with the User. Clean Up a. When complete, close the software by clicking the red X in the upper right corner of the window or clicking on File and selecting Exit from the dropdown menu. b. Open the lid of the LI COR Biosciences Odyssey; a short, soft beeping will be heard to indicate the laser has been turned off. Remove the membrane. c. Clean the scanning surface of the Odyssey using a Kimwipe dampened with distilled water. Dry thoroughly with a Kimwipe, then close the lid. A short, soft beeping will be heard to indicate the laser is ready for the next task. d. Sign out in the Equipment Usage Log as indicated in Section 3.3c. 3.7 Core Materials A brayer is provided on the bench with the LI COR Biosciences Odyssey. Currently, there is also a bottle of distilled water and a box of Kimwipes located near the brayer. 3.8 User Materials Secondary antibodies compatible with the LI COR Biosciences Odyssey for use at the 700 and 800 nm wavelengths may be located and purchased from the below website. These antibodies will allow for the proper and optimal use of the LI COR Biosciences Odyssey. Author: Janice Thompson Page 6 of 9 Approval Date:

7 Users are individually responsible for the compatibility, maintenance and purchase of these materials. Related Products Use Records a. Records of Use All LI COR Biosciences Odyssey use must be recorded. Refer to 3.3c. b. Error Messages / System Issues All error messages and system issues must be relayed to the Equipment Champion and the Pharmacology & Toxicology Core Facility Manager. This information must be relayed on the same day as equipment use. Error messages/system issues must be recorded. Refer to 3.3c Resource Index LI COR Biosciences Odyssey literature and resources for the following items may be found at the link below. a. Operator s Manual (multiple versions) b. Application Protocols Manual c. User Guide (multiple versions) d. Etc. =Manuals&sort=1 4.0 Competences, Authorization and Training New Users must receive proper authorization from either the Equipment Champion and/or Pharmacology & Toxicology Core Facility Manager before equipment use. A new User may contact the Equipment Champion or Pharmacology & Toxicology Core Facility Manager to schedule training. Training includes SOP and instrument familiarization and any additional required or specialized training. Once training is complete, authorization maybe issued and a User Name and Password will be established. All Users are individually responsible for current SOP familiarization. 5.0 SOP Performance and Equipment Review The effectiveness of the SOP B will be monitored by the Pharmacology & Toxicology Core Facility Manager, Equipment Champion and all Users. Any procedural or qualitative deviations will be reflected within an updated SOP. Any approved User should aptly report any procedural or Author: Janice Thompson Page 7 of 9 Approval Date:

8 qualitative issues and/or errors to the Pharmacology & Toxicology Core Facility Manager or Equipment Champion. The Equipment Champion s name and contact information can be found on the Core Facility Identification Sticker. The Core Facility Identification Sticker is located on the LI COR Biosciences Odyssey. The Core Facility Manager s contact information may be found on the Core Facility Contact Posting within room B446. Updated SOPs will be published and approved Users will be notified. SOP: B review will occur every two years. 5.1 Update Date: Reason for SOP Change: New 5.2 Update Date Reason for SOP Change New 6.0 Definitions Any standard defined term specifically used in the Pharmacology and Toxicology Core Facilities SOP. These are terms that carry a unique Core Facility definition that is pertinent to proper SOP understanding and implementation. A list of terms and definitions is maintained to allow Users a quick and efficient reference. Listed below are common Pharmacology and Toxicology Core Facility terms. New common terms are to be approved and added, with definition, to the term reference guide. a. SOP Standard Operating Procedure, which is a standard guide that officially standardizes the process of control, maintenance and ownership of the LI COR Biosciences Odyssey. The SOP number stand for (xxx. xxx. xxx) equipment number. room number. SOP version number. b. Originator / Author The individual representing the Pharmacology and Toxicology Core Facilities that created SOP: B c. Stakeholder Any individual that uses the LI COR Biosciences Odyssey, including the Pharmacology and Toxicology Core Facilities Department. d. New User An individual who has not completed the requirements of section 4.0. e. Approved User An individual who uses or performs the task of which is the subject of the SOP and has fulfilled section 4.0. This title may only be given by the Equipment Champion and / or the Pharmacology and Toxicology Core Facilities Manager. Author: Janice Thompson Page 8 of 9 Approval Date:

9 f. Champion An individual whose direct expertise with the LI COR Biosciences Odyssey has been recognized by the Pharmacology and Toxicology Core Facilities Committee. The Pharmacology and Toxicology Core Facility Committee may only award the champion title. 7.0 Appendix The below signatures and dates are required for full SOP approval and implementation. This SOP was written by: This SOP was reviewed by: Janice Thompson Dr. Stephanie Watts This SOP was authorized by: Dr. J.R. Haywood Issue Date: Author: Janice Thompson Page 9 of 9 Approval Date: