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vitrobot [2023/01/13 14:22] – crucifixvitrobot [2023/11/01 20:18] (current) – external edit 127.0.0.1
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   * fill a dry 5 L tank with liquid nitrogen   * fill a dry 5 L tank with liquid nitrogen
   * label grid boxes with the name of the experiment   * label grid boxes with the name of the experiment
-  * check several things: +  * check plasma cleaner is reserved 
-       * plasma cleaner is reserved+  * Assemble the necessary tools:
        * EM_grids grids are available and present in sufficient number        * EM_grids grids are available and present in sufficient number
-       * all part of the coolant container are dry +       * all part of the coolant container (make sure that they are dry) 
-       * both vitrobot tweezers, big tweezer and blot paper clipping ring are not missing+       * both vitrobot tweezers, big tweezer and blot paper clipping
  
 ==== Starting up the vitrobot ==== ==== Starting up the vitrobot ====
Line 56: Line 56:
   * 1. 'blot total' needs to be '1' for blotting to occur   * 1. 'blot total' needs to be '1' for blotting to occur
   * 2. check 'Humidifier off during process' and 'Skip grid transfer' in the miscellaneous' side bar   * 2. check 'Humidifier off during process' and 'Skip grid transfer' in the miscellaneous' side bar
-  * 3. set your desired blot time, force blot and waiting time (o for unsupported condition ) +  * 3. Optional : set your desired blot time, force blot and waiting time (o for unsupported condition ) 
 +           * all these setting are checked at the begining of the freezing cycle  
                                                                          
 ==== Set humidity and temperature parameters in climate chamber ==== ==== Set humidity and temperature parameters in climate chamber ====
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             * the metallic drawer is **ALWAYS stored in the chamber**             * the metallic drawer is **ALWAYS stored in the chamber**
   * 5. close the door to prevent dust contamination inside the chamber   * 5. close the door to prevent dust contamination inside the chamber
-  * 6. ++ place EM_grids inside the metallic drawer |<div>+  * 6. place EM_grids inside the metallic drawer  
 +          * ++ EM_grids with support film facing up|<div>
       * remove the cover of the metallic drawer by sliding       * remove the cover of the metallic drawer by sliding
       * place EM_grids inside       * place EM_grids inside
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    * 11. if not yet done on vitrobot, enable the humidity switchbox to start      * 11. if not yet done on vitrobot, enable the humidity switchbox to start  
  
-==== Assemble different part of the coolant container like showing below ====+==== Assemble different part of the coolant container as pictured below ====
 {{ :coolant-container-built.jpg?nolink |}} {{ :coolant-container-built.jpg?nolink |}}
  
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       * place EM_grids on the edge of the glass       * place EM_grids on the edge of the glass
 </div>++ </div>++
-  * 10. put the glass slide in a closed crystallizer to transfer safetly grids to vitrobot bench +  * 10. put the glass slide in a closed crystallizer to transfer safely grids to vitrobot bench 
   * 11. load or not new grids in the drawer and close it    * 11. load or not new grids in the drawer and close it 
   * 12. put back in place the closed drawer on the support table   * 12. put back in place the closed drawer on the support table
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             * on the plateform ** NOT on the inlet pump**              * on the plateform ** NOT on the inlet pump** 
                    {{ :support_grid-inchamber8cm.jpg?nolink |}}                    {{ :support_grid-inchamber8cm.jpg?nolink |}}
-   * 9. ++ close the door |<div>+   * 15. ++ close the door |<div>
        * a. be sure the seal is in place and clean (see part.7)         * a. be sure the seal is in place and clean (see part.7) 
        * b. move the lid around its axis to bring it over the opening of the chamber        * b. move the lid around its axis to bring it over the opening of the chamber
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 </div>++  </div>++ 
    * 10. press VACCUM to bring the chamber under vaccum    * 10. press VACCUM to bring the chamber under vaccum
 +           * either grids are waiting ready for an additionnal run of freezing at vitrobot (step 4-to 15)
 +           * or the fishione remains on for next user
 +           * At the end of the day, the fishione can be switch off
  
  
  
- +======Everything is ready for plunge freezing sample:========
- +
- +
-==== Start freezing  ==== +
- +
- +
-       * 1. set or checkcheck the blotting condition +
-       * 1. click on "place new grid" to bring in loading position the plunge rod +
-       * 2. Attach Vitrobot tweezers with a Glow discharged grid onto the plunge rod +
-       * 3. press to bring up the tweezers into the blotting chamber +
-       * 4. place the container onto the ethane lift +
-       * 5. press to lift it +
-       * 6. click on "process" +
-       * 7. apply sample to grid +
-       * 8. click on "continue" to blot and plunge +
-       * 9. carrefully detach tweezers from the plunge rod +
-       * 10. By maintaining the grid inside the ethan cup, place the coolant container on the table  +
-       * 11. transfer grid to the grid position +
-       * 12. Dry tweezers then repeat  +
- +
-* Now vitrobot is ready for freezingbeforeDo it when you are almost ready to freeze to keep the soaking time as reproducible as possible +
- +
-       ====== At this point already is ready for freezing ====== +
- +
-    At this point already is ready for freezing:+
     climate chamber is equilibrated in temperature and humidity.     climate chamber is equilibrated in temperature and humidity.
-    Filter paper are mounted and soaking time is controlled+    Filter paper are mounted 
     ethane is liquefied     ethane is liquefied
-    Grids are loaded and ready to be plasma cleaned+    Grids are freshly plasma cleaned
  
  
-       *  +==== Start freezing at the vitrobot ====
  
-==== Preparation of the grid ==== 
  
 +       * 1. check that the correct freezing parameters are entered 
 +       * 2. click on "place new grid" to bring in loading position the plunge rod
 +       * 3. Lock a Glow discharged grid on tweezers
 +                 * Grasp as little of the grid as possible with the tweezers (but enough that it will not fall out during freezing)
 +                 * ++ Make sure that the black clamping ring is fixed not more down to the first groves. |<div>
 +{{ :rightclamp_twezzers.jpg?nolink |}}
 +</div>++
 +       * 4. then fix the tweezers onto the plunge rod
 +               * with the support film support on the right for right-handers and on the left for left-handers
 +       * 5. press on "continue" to bring up the tweezers into the blotting chamber
 +       * 6. place the container onto the ethane lift
 +               * Always check that ethane is at right temperature  
 +       * 7. press on to lift it
 +       * 8. click on "process"
 +       * 9. apply sample to grid
 +       * 10. click on "continue" to blot and plunge
 +       * 11. carrefully detach tweezers from the plunge rod
 +       * 12. By maintaining the grid inside the ethan cup, place the coolant container on the table 
 +       * 13. transfer grid to the grid position
 +       * 14. Dry tweezers
 +       * 15. then repeat sequence 1-14 for all glow discharged grids
  
- ++3. Bring the vitrobot to hermetically close the chamber|<div> 
-         * do the following dry Run: 
-  * A glow discharge grids is attached to the twezzers 
-  
-==== Start freezing ==== 
-  
  
  
-   
-  * ++ Do it when you are almost ready to freeze to keep the soaking time as reproducible as possible|<div> 
-Decrire 
-</div>++ 
- 
-==== Mounting the filter papers, IMPERATIVELY wear gloves ==== 
- 
-  * ++ Do it when you are almost ready to freeze to keep the soaking time as reproducible as possible|<div> 
-Decrire 
-</div>++ 
            
  
vitrobot.1673619760.txt.gz · Last modified: (external edit)