Daniel:Notebook/RNAFACS/2015-10-1

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Nuceli Adhesion[edit]

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Since the mouse trial (yesterday) went well I am going to try out some human nuclei.

  1. Clean glass slide in 100% EtOH; allow to air dry
  2. Remove cells from -80C and spin down for 5 min at 1000g
    1. Remove supernatant, careful not to disturb the pellet
  3. Add 500uL of 4% paraformaldehyde to the tube and wait 5 min
  4. Centrifuge cells for 5 min at 1000g
    1. Remove supernatant, careful not to disturb the pellet
  5. Add 500 uL of 70% EtOH
  6. Centrifuge cells for 5 min at 1000g
    1. Remove supernatant, careful not to disturb the pellet
  7. Resuspend nuclei in 20 uL 70% EtOH
  8. Deposit on to glass slide
  9. Use a coverslip to gently spread the 20 uL across the slide
    1. Be careful not to scratch the slide
  10. Dry the slide using a heating block at 60C for 10-15 min
  11. Add 15 uL Slowfade + DAPI and seal with a coverslip