Leica EM GP2 Standard Operation Protocol
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Also See for EM GP2:
- User manual (3 pages) ,
- Operating manual (82 pages) ,
- Instructions for use manual (60 pages)
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Summary of Contents for Leica EM GP2
- Page 1 GP2 SOP – CPOS Cryo EM V1.0 2024 Leica EM GP2 Standard Operation Protocol A. Preparation 1) Sign in the logbook. 2) Take out the toolbox from the drawer. 3) Switch on the GP2.
- Page 2 GP2 SOP – CPOS Cryo EM V1.0 2024 4) Turn on the Touch screen to initialize the system. 5) Connect syringe (with 90mL distilled water) to the tube with the Luer™ lock and open the valve. Add water into the humidifier tank mounted on the right-hand side of the environmental chamber by pressing the syringe piston slowly but steadily to fill in the water.
- Page 3 GP2 SOP – CPOS Cryo EM V1.0 2024 When the filling of the humidifier tank falls below its minimum level during operation, the system will beep and indicate that refilling is needed. Re-filling requires only 20 ml distilled water. 7) Pull out the drip tray to the right, discard of the condensate, and re-insert it to the original position. 8) Mount fresh filter paper (Whatman™...
- Page 4 GP2 SOP – CPOS Cryo EM V1.0 2024 9) After setting in the fresh filter paper, press the button on the main screen to reset the counter. The instrument will rotate one ring of filter paper 10 times before the paper needs to be replaced. The number of blots remaining is displayed on the main screen and can be reset by pressing the button.
- Page 5 GP2 SOP – CPOS Cryo EM V1.0 2024 12) Add LN to the freezing chamber until just above the metal mesh. It will take around 1.8 litres LN cool the Dewar and fill it to 100%. A warning will be displayed when the LN flow cannot function anymore below 25% LN level.
- Page 6 GP2 SOP – CPOS Cryo EM V1.0 2024 14) Open the Main value 1 on the ethane tank. Slowly adjust the flow rate via turning the Flow valve 2. Check the ethane level continuously looking through the window of the liquefier. 15) When the ethane container is filled, close all valves on the ethane bottle.
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Page 7: Program Settings
GP2 SOP – CPOS Cryo EM V1.0 2024 C. Program settings 1) Environment Parameters a) Press “Set” to change the current value of chamber temperature, chamber relative humidity, window heater, cryogen temperature and GN flow on the keypad and “OK” to confirm. 2) Load Specimen Parameters The “Load Specimen”... - Page 8 GP2 SOP – CPOS Cryo EM V1.0 2024 b) For right-handed operators and front-side blotting: c) For left-handed operators and back-side blotting: d) For left-handed operators and front-side blotting: e) A delay between specimen application and the actual filter paper blotting (i.e. pre-blotting time) can be set with the “Delay time before blotting –...
- Page 9 GP2 SOP – CPOS Cryo EM V1.0 2024 3) Blot Parameters a) Single Blotting without Sensor In this mode, the filter paper will advance to the user calibrated position of the grid for blotting once, remain there for a defined time, retract, and then either proceed with the freezing process automatically or wait for the user to initiate freezing.
- Page 10 GP2 SOP – CPOS Cryo EM V1.0 2024 b) Single Blotting with Sensor This is the most automatic setting, intended to increase reproducibility: around the expected position of the grid, the advancement of the filter paper will slow down, and a blotting sensor will constantly check for the point where the filter paper is getting wet and starts blotting the sample.
- Page 11 GP2 SOP – CPOS Cryo EM V1.0 2024 c) Multiple blotting This mode of blotting is preferable for viscous specimens that cannot be thinned efficiently by single side blotting. * The blotting sensor cannot be used in conjunction with multiple blotting In this mode, it is essential to have the horizontal position of the filter paper for blotting calibrated accurately for both the home position and the 180°...
- Page 12 GP2 SOP – CPOS Cryo EM V1.0 2024 • Skip transfer position / Automatically move to transfer position: Entirely depending on the user’s personal preference, the EM GP2 allows to: “Skip transfer position” “Automatically move to transfer position” ✓ ...
- Page 13 GP2 SOP – CPOS Cryo EM V1.0 2024 D. Freezing a grid 1) Hold the grid with the GP2 tweezers. Fix the black locking sleeve to the first groove on the tweezers. Make sure to orientate the desired side of the grid on the side where you are going to load your sample (e.g.
- Page 14 GP2 SOP – CPOS Cryo EM V1.0 2024 4) Click “LOAD SPECIMEN”. The chamber will lower and if a 180° rotation for loading has been set up, the forceps will rotate by 180°. 5) Open the side port of the environmental chamber and dispense a 3 - 5 µl specimen onto the grid. Close the side port immediately to maintain a stable atmosphere.
- Page 15 GP2 SOP – CPOS Cryo EM V1.0 2024 7) If “automatically plunge after blotting” is selected in the parameters, the GP2 will automatically plunge the grid into the secondary cryogen after blotting (and the elapse of the optional post-blot delay). If this option is not enabled, the user must press the “PLUNGE”...
- Page 16 GP2 SOP – CPOS Cryo EM V1.0 2024 10) Remove the lid of the grid box transfer container. Set the grid box to an open position. Remove the forceps from the gantry tilting it out forwards, do not let the frozen grid touch the wall of the ethane container.
- Page 17 GP2 SOP – CPOS Cryo EM V1.0 2024 E. Bake out and shut down 1) Remove any remaining water from the humidifier tank by aspirating it with the syringe. 2) Remove filter paper and leave the door of the environmental chamber open. 3) Remove ethane container.
- Page 18 GP2 SOP – CPOS Cryo EM V1.0 2024 5) After the bake out is complete, the environmental chamber will move automatically to the bottom position. Wait until the chamber arrives at the lowest position. Then, switch off the main switch at the back.
- Page 19 GP2 SOP – CPOS Cryo EM V1.0 2024 Checklist before leaving the lab: • Turn off the ethane tank to prevent any potential leaks or accidents. • Shut down the GP2 following the proper procedures. • Remove the filter paper and dispose of it in the designated waste bin. •...
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