Daniel:Notebook/HiResChrPaint/2014-1-22

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FISH[edit]

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Prepping some samples to hopefully use with Alu positive control by the weekend.

FISH Dish Preparation[edit]

1. Prepare a cell suspension of 500 000-2 000 000 cells/mL in growth media
2. Add 100 uL of cell suspension per slide roughly in the center
3. Allow cells to adhere for 1-3 hours at the growth temp of the cell line
4. Rinse slides briefly in 1X PBS
5. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
6. Rinse briefly in 1X PBS
7. Incubate for 5 minutes in 2X SSCT
8. Incubate for 5 minutes in 2X SSCT + 50% (v/v) formamide
9. Replace with fresh 2X SSCT + 50% (v/v) formamide for storage at 4C
10. Slides are typically best used within 1-2 weeks of creation
  • For cancer cell line (NCIH 1975) I'd recommend ~10 uL cell suspension and 90 uL DMEM

Alu Control (Started 01/08/2014)[edit]

Dye Coupling[edit]

Testing 3 samples:

  • Blank (Just water)
  • Cot1-A Ethanol precipitating 1 ug of Cot1 DNA just prior to dye labeling, as suggested;
0-1 1 uL Cot1 DNA, 49 uL nfH2O, 5 uL 3M NaOAc, 120 uL 100% EtOH; 
0-2 Store at -80C for 30 minutes
0-3 Centrifuge at 4C for 15 minutes at 12000 rpm
0-4 Remove liquid and replace with 100 uL 70% EtOH
0-5 Centrifuge at 4C for 10 minutes at 12000 rpm
0-6 Remove liquid and allow to air dry
  • Cot1-B Vacuum drying 1 ug of Cot1 DNA, it is the easier method for dehydrating that I usually go with
  • DNA only, add 1 uL (1 ug);

After samples dried, left overnight at 4 C. Continued tomorrow