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vitrobot [2023/01/13 11:53] – 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) 
-       * close climate chamber's door if open +       * both vitrobot tweezers, big tweezer and blot paper clipping
-       * Paper filter clipping ring are not missing+
  
 ==== Starting up the vitrobot ==== ==== Starting up the vitrobot ====
  
-  * ++1. Switching on vitrobot|<div>+  * 1. close climate chamber's door if open 
 +  * ++2. Switching on vitrobot|<div>
   * Press the hard lock switch on the right backside of the machine   * Press the hard lock switch on the right backside of the machine
 {{ :swith-on-vitrobot.jpg?nolink |}} {{ :swith-on-vitrobot.jpg?nolink |}}
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-  * 2. Make sure the humidifier is operational+  * 3. Make sure the humidifier is operational
        * ++ check humificator beaker state in humidity panel|<div>        * ++ check humificator beaker state in humidity panel|<div>
 {{ :humidify-beaber-state2.jpg?nolink |}} {{ :humidify-beaber-state2.jpg?nolink |}}
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        * ++ if EMPTY refill it with distilled water |<div>        * ++ if EMPTY refill it with distilled water |<div>
 **Use ONLY distilled water ** **Use ONLY distilled water **
-     * 1. Fill a syringe with 60 ml distilled water +     * a. Fill a syringe with 60 ml distilled water 
-     * 2. inject the whole volume through the plastic tube at the bottom part of the humidifier +     * b. inject the whole volume through the plastic tube at the bottom part of the humidifier 
-     * 3. When removing the syringe from the tube, be careful not to tear it from its location+     * c. When removing the syringe from the tube, be careful not to tear it from its location
  
 {{ :capture_fill_humidif.jpg?nolink |}} {{ :capture_fill_humidif.jpg?nolink |}}
 </div>++ </div>++
  
-  * ++3. hermetically close the chamber|<div>+  * ++4. hermetically close the chamber|<div>
          * do the following dry Run:          * do the following dry Run:
              * Place new grid / continue / place ethane container              * Place new grid / continue / place ethane container
 </div>++ </div>++
         * the white latch remains in open position after swith on the power.           * the white latch remains in open position after swith on the power.  
 +
 +
  
 ==== Set blotting process parameters ==== ==== Set blotting process parameters ====
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   * 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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                       {{ :capture2_adjust-hum.jpg?nolink |}}                       {{ :capture2_adjust-hum.jpg?nolink |}}
 </div>++ </div>++
-    * 3. wait until desired temperature is reached +    * ++3. wait until desired temperature is reached then enable the humidity switchbox to start |<div>
-           * ++ take advantages from this waiting time to start next step :|<div> +
-==== load grids inside the plasma cleaner ==== +
-</div>++ +
-    * ++4. then enable the humidity switchbox to start |<div>+
             * a. read current temperature (red value) in panel temperature             * a. read current temperature (red value) in panel temperature
             * b. press ON/OFF switch in panel humidity             * b. press ON/OFF switch in panel humidity
 {{ :swithon-humidif.jpg?nolink |}} {{ :swithon-humidif.jpg?nolink |}}
 </div>++ </div>++
 +           * take advantages from this waiting time to do the two following steps 
 +                       * Mounting the filter papers
 +                       * load grids inside the plasma cleaner
 +
 +==== Mounting the filter papers ====
 +
 +  * IMPORTANT **always wears gloves**
 +  * place blot papers onto the blotting pads with clipping ring
 +  * support the blotting arms during assembling to prevent damage to the arms
 +
 +
  
 ==== load grids inside the plasma cleaner ==== ==== load grids inside the plasma cleaner ====
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   * ++ 3. Open chamber and make sure the seal remains in place |<div>   * ++ 3. Open chamber and make sure the seal remains in place |<div>
 +{{ :open_chamber-stepbystep3.jpg?nolink |}}
        * a. unlock the rod by pushing the button on it        * a. unlock the rod by pushing the button on it
        * b. pull the rod upwards to lift the cover          * b. pull the rod upwards to lift the cover  
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              * It can either stick on the cover or come out of its channel              * It can either stick on the cover or come out of its channel
 </div>++ </div>++
-                         {{ :open_chamber-stepbystep.jpg?nolink |}}+
   * 4. ++ retrieve the metallic drawer and place it on the table |<div>   * 4. ++ retrieve the metallic drawer and place it on the table |<div>
   * the support carrying the drawer can be taken out by lifting it by the rod.   * the support carrying the drawer can be taken out by lifting it by the rod.
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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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   * 4. wait 10 to 15 min for system to equilibrate   * 4. wait 10 to 15 min for system to equilibrate
   * ++ 5. then liquefy the ethan in the central cup already cooled to liquid nitrogen temperature |<div>   * ++ 5. then liquefy the ethan in the central cup already cooled to liquid nitrogen temperature |<div>
-Ethan is liquefied by flowing a stream of gas through plastic tubing connected to an ethane flask into     the central cup +             * Ethan is liquefied by flowing a stream of gas through plastic tubing connected to an ethane flask into the central cup 
 </div>++ </div>++
   * ++ 6. let ethane start to solidify for few seconds|<div>   * ++ 6. let ethane start to solidify for few seconds|<div>
-apparence of a white solid rim around the wall container indicates that ethane starts to solidify+             * apparence of a white solid rim around the wall container indicates that ethane starts to solidify
 </div>++ </div>++
   * ++ 7. then remove the spindle (C)|<div>    * ++ 7. then remove the spindle (C)|<div> 
-solidified ethan makes stick together the spindle and the ethan cup. +            * Solidified ethan makes stick together the spindle and the ethan cup. 
-Thawing the frozen ethan by placing the second ethan cup on the spindle for 10sec is the more careful way to remove it+            * Thawing the frozen ethan by placing the second ethan cup on the spindle for 10sec is the more careful way to remove it with a big tweezer
 </div>++ </div>++
   * 8. place grid box into the grid box platform (B)   * 8. place grid box into the grid box platform (B)
           * make sure that the grid box is labelled with the experiment name           * make sure that the grid box is labelled with the experiment name
- 
-==== Mounting the filter papers ==== 
- 
-  * IMPORTANT **always wears gloves** 
-  * place blot papers onto the blotting pads with clipping ring 
-  * support the blotting arms during assembling to prevent damage to the arms 
- 
  
  
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        * The gas plasma generated can be seen through the window as a pink light        * The gas plasma generated can be seen through the window as a pink light
        * remaining time can be followed in the time display        * remaining time can be followed in the time display
-  * 5. Retrieve the drawer +  * 5. press Vent Lock to bring the chamber under atmospheric pressure 
-  * 6. place the plasma cleaned grids on a glass slide+       * State is reached when message Lock At Atmosphere appears 
 +  * 6. Open the chamber 
 +  * ++ 7. Retrieve the drawer and place it on the table |<div> 
 +       * the support carrying the drawer can be taken out by lifting it by the rod. 
 +       * Be sure to keep the table horizontal so as not to slide the drawer 
 +</div>++ 
 +  * 8. close the door to prevent dust contamination inside the chamber 
 +  * 9. ++ place the plasma cleaned grids on a glass slide  |<div> 
 +      * remove the cover of the metallic drawer by sliding 
 +      * place EM_grids on the edge of the glass 
 +</div>++ 
 +  * 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  
 +  * 12. put back in place the closed drawer on the support table 
 +  * 13. ++ open the door and make sure the seal remains in place|<div> 
 +      * a. if necessary put the seal back in place 
 +         * It can either stick to the cover or come out of its channel 
 +      * b.clean it just by passing your finger 
 +</div>++ 
 +   * 14. put back in place the metallic drawer inside the chamber 
 +            * on the plateform ** NOT on the inlet pump**  
 +                   {{ :support_grid-inchamber8cm.jpg?nolink |}} 
 +   * 15. ++ close the door |<div> 
 +       * 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 
 +       * c. unlock the rod by pushing the button 
 +       * d. gently allow the lid to slide down   
 +</div>++  
 +   * 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
  
- 
-==== Set blotting process parameters ==== 
  
  
-==== Set freezing condition ==== +======Everything is ready for plunge freezing sample:========
- +
- +
-       * 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.1673610832.txt.gz · Last modified: (external edit)