Documentation:Standard Operating Procedures for IEF-PAGE using PhastSystem

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  1. Please obtain proper training from the FNH education and research support technician Imelda Cheung (Imelda.Cheung@ubc.ca) prior to using the PhastSystem.
  2. Put a saran wrap on the bench to prevent spills of solutions to make marks on the table.
  3. Turn on the machine (button at the back of the system).
  4. Set up machine: Press standby temperature and set T=15oC then press DO.
  5. Put parafilm (with the white piece on) on sample holder (black) to cover the circles.
  6. Take the IEF 3-9 gels from the cold room/fridge.
  7. Clean the apparatus with ddH2O and wipe with kimwipes.
  8. Only use the right side if only running one gel.
  9. Put 100 uL of ddH2O onto the bed.
  10. Cut open the gel and carefully use the forcep to open the gel. Check that the plastic cover is on top, then bend the tab upward to recognize which side is up.
  11. Put the gel on the bed and ensure that no air bubbles are trapped.
  12. Remove excess ddH2O by touching the edges with kimwipes.
  13. Remove the top plastic cover of the gel (Keep the cover for later use).
  14. Lower the electrode and if the electrode does not lie flat on gel surface, move the red knob downwards (the higher position is for SDS-PAGE).
  15. Lower the applicator holder and put down the lid.
  16. Remove the white piece of the parafilm on the sample holder.
  17. Start loading samples and the marker onto the sample holder (5 uL each) and record which sample you have put in which lane.
    • Note: make sure no air bubbles are observed otherwise in the next step you may not obtain the proper amount of sample.
  18. Open the 8/1 sample applicator (i.e. 1 ul each for 8 lanes), gently touch the wells of the sample applicator to obtain samples.
  19. Gently put the comb in the middle slot. Make sure we do not touch the comb and press it down.
  20. Press SEP start, then 1 or 2 gels, then DO, then 3. IEF 3-9 and DO.
  21. Monitor the gel progress (around 15-30 minutes).
  22. When the gel is done, the machine will make a beeping sound. Stop the gel by pressing SEP start.
  23. Throw away the sample applicator.
  24. Take the gel out and immediately put it into the staining solution to stain.
  25. Clean the apparatus with ddH2O and wipe with kimwipes.
  26. Turn off machine and sign the logbook.
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