Daniel:Notebook/HiResChrPaint/2014-7-17
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Strand Control (Started 07/08/2014)[edit]
Hybridization (from 7/15/2014)[edit]
- Remove coverslip carefully and wash in pre-warmed 2X SSCT at 60C for 15 minutes
- Incubate in 2X SSCT at room temperature for 10 minutes
- Incubate in 0.2X SSC at room temperature for 10 minutes
- Dry with kimwipes; do not let the surface containing the cells touch the kimwipes
- Add 15 uL DRAQ5 (diluted 1:1000) to center of dish
- Cover with 22x22 #1.5 coverslip; seal with nail polish
- Allow at least 30 minutes for nail polish to dry
- Slides are quite stable for several months at 4C
Slide Preparation[edit]
- Prepare a cell suspension of 500 000-2 000 000 cells/mL in RPMI
- Detach cells with 0.5mL trypsin
- Resuspend with 9.5 mL media
- Add 50 uL of cell suspension per dish to the cener; add 50 uL RPMI
- Allow cells to adhere for 3.5 hours at 37 C
- Rinse slides briefly in 1X PBS
- Fix slides for 10 minutes in 4% (v/v) paraformaldehyde in 1X PBS
- Rinse briefly in 1X PBS
- Incubate for 5 minutes in 1 mL of 2X SSCT
- Incubate for 5 minutes in 1 mL of 2X SSCT + 50% (v/v) formamide
- Remove media and add 1 mL of 2X SSCT + 50% (v/v) formamide for storage at 4C; wrap with parafilm
- Slides are typically best used within 1-2 weeks of creation