Daniel:Notebook/HiResChrPaint/2014-7-17

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Strand Control (Started 07/08/2014)[edit]

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Hybridization (from 7/15/2014)[edit]

  1. Remove coverslip carefully and wash in pre-warmed 2X SSCT at 60C for 15 minutes
  2. Incubate in 2X SSCT at room temperature for 10 minutes
  3. Incubate in 0.2X SSC at room temperature for 10 minutes
  4. Dry with kimwipes; do not let the surface containing the cells touch the kimwipes
  5. Add 15 uL DRAQ5 (diluted 1:1000) to center of dish
  6. Cover with 22x22 #1.5 coverslip; seal with nail polish
    1. Allow at least 30 minutes for nail polish to dry
    2. Slides are quite stable for several months at 4C

Slide Preparation[edit]

  1. Prepare a cell suspension of 500 000-2 000 000 cells/mL in RPMI
    1. Detach cells with 0.5mL trypsin
    2. Resuspend with 9.5 mL media
  2. Add 50 uL of cell suspension per dish to the cener; add 50 uL RPMI
  3. Allow cells to adhere for 3.5 hours at 37 C
  4. Rinse slides briefly in 1X PBS
  5. Fix slides for 10 minutes in 4% (v/v) paraformaldehyde in 1X PBS
  6. Rinse briefly in 1X PBS
  7. Incubate for 5 minutes in 1 mL of 2X SSCT
  8. Incubate for 5 minutes in 1 mL of 2X SSCT + 50% (v/v) formamide
  9. Remove media and add 1 mL of 2X SSCT + 50% (v/v) formamide for storage at 4C; wrap with parafilm
  10. Slides are typically best used within 1-2 weeks of creation