Daniel:Notebook/HiResChrPaint/2014-1-22
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FISH[edit]
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]
- Following ULYSIS Protocol
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